Twist signaling inhibitor compositions and methods of using the same

ABSTRACT

The present invention relates to compositions comprising TWIST signaling inhibitors and optionally one or more anti-cancer agents, and methods of using the compositions for the treatment of cancer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the priority and the benefit of U.S. ProvisionalApplication No. 62/294,229, filed Feb. 11, 2016, and U.S. ProvisionalApplication No. 62/425,143, filed Nov. 22, 2016, the content of each ofwhich is incorporated herein by reference in its entirety and for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant NumbersP30CA33572 and CA133697 awarded by the National Institutes of Health.The Government has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 48440-614001US_ST25.TXT, createdFeb. 10, 2017, 10,246 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND

Breast cancer is the second leading cause of cancer deaths in women inthe US with over 246,660 new diagnoses expected in 2016 and almost40,450 deaths. The “triple-negative breast cancer” (TNBC) (estrogenreceptor [ER]-negative, progesterone receptor [PR]-negative, and humanepidermal growth factor receptor 2 [HER2]-negative) poses a particularlydifficult challenge, patients with these tumors have poor prognosisbecause of inherent resistance to therapy and a high incidence ofmetastasis. There is thus a critical need for novel efficacioustherapies for patients with TNBC. Provided herein are solutions to theseand other problems in the art.

SUMMARY

In a first aspect, there is provided a composition including a TWISTsignaling inhibitor bound to a delivery vehicle.

In another aspect, there is provided an siRNA including a sequence ofany one of SEQ ID NOs: 1-12.

In another aspect, there is provided a DNA sequence encoding an siRNAsequence including a sequence of any one of SEQ ID Nos: 1-10.

In another aspect, there is provided a method of reversing resistance toan anti-cancer drug in a subject. The method includes administering atherapeutically effective amount of a TWIST signaling inhibitor to thesubject.

In another aspect, there is provided a method of treating cancer in asubject in need thereof. The method includes administering to thesubject a therapeutically effective amount of a TWIST signalinginhibitor.

In another aspect, there is provided a method of inhibiting metastasisin a subject. The method includes administering a therapeuticallyeffective amount of a TWIST signaling inhibitor to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D. siRNA-MSN complexes for use in vitro and in vivo. FIG.1A. Individual pores are visible in TEM micrographs of ˜100 nm MSNs.FIG. 1B. Particle size within a batch is uniform. FIG. 1C. Schematic ofsiRNA silencing via RNA induced silencing complex (RISC). siRNA contains2-OMe-U and inverted abasic ribose modifications, to decreasedegradation and immune stimulation, and to guarantee the guide strand isloaded into RISC. TWIST1 siRNA binds to TWIST1 mRNA, leading to itscleavage by Ago2. The siRNA guide strand is reused to recognizeadditional TWIST1 mRNAs. FIG. 1D. Sequences of si419 showing positionsof 2-OMe and inverted abasic modifications. Sequences: siTw-419HPassenger (SEQ ID NO:11); siTw-419H Guide (SEQ ID NO:12).

FIGS. 2A to 2D. siRNA enters cells and knocks down TWIST1 in vitro. FIG.2A. TWIST1 siRNA delivered using LIPOFECTAMINE® 2000 decreases levels ofTWIST1 protein up to 72 hours following transfection. FIG. 2B. i. Mergedconfocal image of GFP (grey), DAPI (white dots) and siRNA (bright whitedots). ii. Magnification of cell from center right of i. siRNA showsexpected perinuclear localization. iii-v. Single color images. FIG. 2C.Delivery of TWIST1 siRNA using MSNs produces significant knockdown at 72hours post transfection, but TWIST1 protein levels stabilize after oneweek. FIG. 2D. qRT-PCR data demonstrate that TWIST1 mRNA levels are alsoreduced 72 hours following MSN+siRNA treatment.

FIGS. 3A to 3B. TWIST1 knockdown reduces downstream pathways in vitro.FIG. 3A. MDA-MB-435S cells with TWIST1 knocked down are slower tomigrate and seal a scratch wound. FIG. 3B. ELISA reveals thatMDA-MB-435S cells treated with TWIST1 siRNA secrete significantly lessIL-8 than nontransfected cells or those transfected with irrelevantcontrol (siGFP).

FIGS. 4A to 4E. MSN+siRNA therapy reduces TWIST expression and tumorgrowth in vivo. FIG. 4A. Gross tumor images reveal smaller, lessvascularized tumors in mice treated with si419H than in control mice orthose given non-chemically-modified si494. Representative images shown.FIG. 4B. si419H treated tumors show a significant drop in weightcompared to untreated or si494-treated tumors. FIGS. 4C-4E. qPCR resultsfrom tumors collected at necropsy. Tumors exhibit loss of TWIST1 (FIG.4C) and its target genes Vimentin (FIG. 4D) and CCL2 (FIG. 4E). In eachcase, more robust knockdown is observed for si419H than si494.

FIG. 5. MTT assay demonstrates that at concentrations used fortransfection in vitro (1 Tx) siRNA-MSN complexes are not inherentlycytotoxic, and do not show appreciable cell death until dose isincreased five-fold. This phenomenon was independent of siRNA sequence.

FIG. 6. Chemical modification of si419 does not significantly changeknockdown efficiency. Unmodified si419 and hybrid si419 containing onlythe passenger strand modifications (H) each show approximately 80%knockdown. Therefore, si419 hybrid was used throughout this work.

FIGS. 7A to 7C. MDA-MB-435S cells express GFP and firefly luciferase.FIG. 7A. Phase contrast and FIG. 7B. green channel images of MDA-MB-435Scells. FIG. 7C. Expression of firefly luciferase is evidenced by strongsignal following D-luciferin injection in mice (Xenogen IVIS, STARR).

FIGS. 8A to 8B. MDA-MB-435S cells metastasized to the lungs in mice.FIG. 8A. MDA-MB-435 cells stain positive for GFP in GFP negative lungtissue. FIG. 8B. Metastases were grouped into one of four categories onthe basis of cell number. No inj, no injection.

FIGS. 9A to 9B. RNA-seq performed on SUM1315 triple negative breastcancer cells stably expressing shRNA against TWIST or control (shScram).FIG. 9A. TWIST1 expression is reduced by sh419 and sh494. FIG. 9B.TWIST2 is only knocked down by sh419. FPKM, fragments per kilobase permillion fragments read.

FIG. 10. Mechanism of dendrimer-mediated siRNA delivery and TWIST1knockdown. 1. siRNA adheres to positive charges on the YTZ3-15dendrimer, and the dendrimer-siRNA complex is administered to tumorcells. 2. Complexes are taken up via macropinocytosis. 3. Complexes aretrafficked to late endosomes. 4. Due to the proton sponge effect,electrostatic interactions between the dendrimer and siRNA are disruptedand siRNA escapes from the disrupted endosome into the cytosol. 5. Oncein the cytosol, siRNA recruits the endogenous RNAi machinery to degradeTWIST1 mRNA. siRNA guide strand is conserved, and the RISC complex isfree to recognize and degrade subsequent TWIST1 mRNAs.

FIGS. 11A to 11C. Stable shRNA-mediated TWIST1 knockdown in SUM 1315.FIG. 11A. Western blot demonstrated robust TWIST1 knockdown in bothshTwistA and shTwistB lines. FIG. 11B. qRT-PCR confirmed TWIST1knockdown at the mRNA level for both stable knockdown lines. FIG. 11C.SUM 1315 cells expressing shTwistA or shTwistB exhibited decreaseddirectional migration compared to those expressing shScram control inwound healing assays. Dashed lines indicate migratory front. Imagesshown are representative data from experiments performed in triplicate.

FIGS. 12A to 12C. YTZ3-15 efficiently delivers siRNA to SUM 1315 cells.FIG. 12A. Left: non-transfected SUM 1315 cells had low backgroundfluorescence. Right: Greater than 99% of YTZ3-15 transfected cells werepositive for AlexaFluor-647 labeled siQ. FIG. 12B. Fluorescentmicroscopy revealed that AlexaFluor-488 labeled siQ was taken up intocells within one day, and AlexaFluor signal was still detectable incells at seven days post-transfection. FIG. 12C. Confocal images of SUM1315 cells stably expressing GFP and transiently transfected withAlexaFluor-647 labeled siQ using YTZ3-15. Lysotracker dye revealed thatsiQ colocalized with late endosomes and lysosomes after incubation withYTZ3-15 siRNA complexes.

FIGS. 13A to 13D. TWIST1 knockdown following YTZ3-15 delivery of siTwistdecreases cell motility and downstream EMT marker expression. FIG. 13A.Compared to siQ control (at seven days), siTwistA (TwA) and siTwistB(TwB) delivered via YTZ3-15 produced >90% knockdown at the mRNA level.Knockdown lasted seven days post-transfection. FIG. 13B. Compared to siQcontrol (at seven days), TwA and TwB delivered via YTZ3-15 producedknockdown of the TWIST1 targets N-Cadherin and Vimentin. N-Cadherin mRNAlevels decreased by >40% after one day, and by approximately 90% afterseven days. Vimentin mRNA was nearly undetectable after one day, andremained at <10% after seven days. FIG. 13C. YTZ3-15 transfection ofsiTwistA decreased directional migration compared to siQ transfectedcells (control) in wound healing assays. Dashed lines indicate migratoryfront. Images shown are representative data from experiments performedin duplicate. FIG. 13D. Top: YTZ3-15 transfection of TwA or TwB resultedin >50% decrease in invasion of SUM 1315 cells through matrigel. Cellswere allowed to migrate for one day, following one day incubation withYTZ3-15-siRNA complexes. Five fields per condition were imaged(representative images shown). Bottom: Quantification of image data.Data are average of five fields per condition. Error bars representstandard deviation.

FIGS. 14A to 14B. YTZ3-15 concentrates in orthotopic breast cancertumors in vivo. FIG. 14A. Representative animals from the mice thatreceived YTZ3-15+siQ via intratumoral (IT) and intravenous (IV)injections. Control animals received IV injections of the dendrimercomplex but had no tumors. FIG. 14B. Ex vivo imaging of spleen, kidney,liver and tumors from the three animals shown in FIG. 14A demonstratingconcentration of YTZ3-15+siQ complex in the tumors but not in otherorgans. The units for the scale bars in this figure are p/sec/cm²/sr.

FIGS. 15A to 15E. FIG. 15A. TWIST schematic showing the basic DNAbinding domain, helix-loop-helix dimerization motif, and C-terminalprotein-binding WR domain. FIG. 15B. Reactivation of TWIST in cancersinduces an epithelial to mesenchymal transition (EMT), which in turn hasbeen shown to lead to metastasis and acquired drug resistance. FIG. 15C.Sequences of two TWIST siRNAs targeting the coding region of TWIST mRNA(from top to bottom: SEQ ID Nos 2, 1, 4, 3, respectively). FIG. 15D.Validation of siRNA. Lipofectamine 2000 was used to transfect A2780Rcells with si419 or si494. Western blot reveals robust TWIST knockdownover three days post transfection. shRNA targeting TWIST is shown as apositive control for knockdown. FIG. 15E. Without a carrier, no siRNAenters target cells.

FIGS. 16A to 16E. FIG. 16A. YTZ3-15 PAMAM dendrimer used in thesestudies. Lipid tails encourage formation of micelle structures oncedendrimers are complexed with siRNA (FIG. 16B), which is bound byterminal amines. FIG. 16C. siQ control siRNA tagged with ALEXAFLUOR® 647is efficiently taken up by A2780R and Ovcar8 cells. Scale bar, 100 rpm.FIG. 16D. Both TWIST siRNAs, when delivered using YTZ3-15, result inknockdown of TWIST in A2780R cells, but even 100 nM siRNA produces onlyminimal knockdown in Ovcar8. FIG. 16E. SRB assay reveals that A2780Rcells are sensitized to cisplatin following treatment with YTZ3-15-si494complexes. IC₅₀ is reduced from ˜200 to ˜20 μM.

FIGS. 17A to 17D. FIG. 17A. Schematic of an MSN with pore structure(white). MSNs used in these studies have a PEI coating (purple layer)which binds siRNA (orange). Monomer structure for PEI is shown below.FIG. 17B. Transmission electron micrograph of MSNs. Particles are ofuniform diameter, ˜120 nm. FIG. 17C. A2780R and Ovcar8 cells efficientlytake up MSNs loaded with siQ-AlexaFluor-647. Scale bar, 100 μm. FIG.17D. In both cell lines, si419 and si494 loaded onto MSNs produce robustTWIST knockdown lasting one week post transfection, although TWISTprotein remains at 24 hours post transfection.

FIGS. 18A to 18D. FIG. 18A. In preparation for in vivo studies, si419was chemically modified to include 2′-O-methyluracil and inverted abasicribose caps on the passenger strand. This siRNA is termed si419H.Sequences: si419H Passenger (SEQ ID NO:11); si419H Guide (SEQ ID NO:12).FIG. 18B. Confocal microscopy demonstrates MSN delivery of siRNA toOvcar8-IP cells. siQ-AlexaFluor-647 colocalizes with lysosomes and lateendosomes, as stained by LysoTracker, reflecting proper trafficking ofMSNs to allow siRNA release. FIG. 18C. Western blot confirms that si419Hknocks down TWIST in Ovcar8-IP cells, including at the 24 hourtimepoint. Knockdown is still effective one week post treatment. FIG.18D. SRB cell survival assay reveals that TWIST knockdown sensitizesOvcar8-IP cells to cisplatin. si419H performs similarly to unmodifiedsi419. Ovcar8 is more sensitive than A2780R, hence a less pronouncedeffect of TWIST on drug response in Ovcar8-IP.

FIGS. 19A to 19F. FIGS. 19A, 19C, 19E. Necropsy images of mice treatedwith siQ-AlexaFluor-647 on consecutive days reveal localization ofMSN-siRNA complexes to the tumor. Bright field is shown in FIG. 19A. GFPfluorescence (FIG. 19C) shows all Ovcar8-IP tumor cells within theabdominal cavity. siQ-AlexaFluor-647 fluorescence (FIG. 19E) isconcentrated in the large disseminated mass located near the stomach.FIGS. 19B, 19D, 19F. Imaging of individual organs reveals thatnegligible quantities of MSNs are found in the uterus, liver, kidney, orspleen. Bright field is shown in FIG. 19B. GFP fluorescence image isshown in FIG. 19D. siQ-AlexaFluor-647 fluorescence (FIG. 19F) is mostlyfound in disseminated tumors, including a lesion on the liver surface,with limited signal from the primary tumor. Units for luminescence arephotons/sec/cm²/steradian.

FIGS. 20A to 20B. FIG. 20A. Bioluminescence imaging of Ovcar8-IP tumors.Tumors treated with cisplatin plus MSN-siQ emit noticeably weaker signalthan MSN-siQ only control mice, while those treated with cisplatin plusMSN-si419H exhibit a further loss of signal. FIG. 20B. Quantification ofbioluminescence for all four weeks of treatment as depicted in A forweeks 1 and 4. Units for luminescence are photons/sec/cm²/steradian.

FIGS. 21A to 21C. FIG. 21A. Mice treated with MSN-siQ only have greaternumbers and sizes of tumors than TWIST knockdown mice. Cisplatintreatment eliminated much of the tumor mass, but a combination ofcisplatin and TWIST knockdown yielded the cleanest peritoneal cavity atthe conclusion of the experiment. Arrows indicate tumor foci. Onerepresentative image shown per group (n=4). FIG. 21B. Quantification ofnumbers of disseminated masses seen in images of mice, as seen in A.Cisplatin treatment reduced metastasis incidence by approximately 50%,while combination of cisplatin with MSN-si419H reduced this 75%. FIG.21C. Quantification of tumor weight for disseminated masses only (left)and total tumor including primary in ovaries (right). Cisplatin, with orwithout TWIST knockdown, produced a significant drop in tumor weight.Addition of si419H led to a significant decrease in disseminated tumormass (p=0.0084), and a drop in total tumor mass as well (p=0.1183).si419 alone did not produce a statistically significant change in tumorweight.

FIGS. 22A to 22D. TWIST overexpression leads to cisplatin resistance andenhanced tumour cell engraftment. FIG. 22A. Western blottingdemonstrates differential expression of TWIST between Ov8GFP stablytransfected cell lines. Blots cropped for clarity; full blots are shownin FIG. 30. FIG. 22B. SRB assay demonstrates that TWIST1 expressionleads to increased survival following exposure to cisplatin,particularly at lower doses (5, 10, and 20 μM, p<0.0001; 40 μM,p=0.0002). FIG. 22C. Time lapse microscopy shows that across two logs ofcisplatin doses, TWIST1 expression leads to faster growth of cells.TWIST1-expressing cells achieve greater confluence over time than TWIST1knockdown cells at corresponding drug dose. Average slopes of the linesindicate a faster rate of growth for TWIST1 cells than sh492 cells untilconfluence is reached. Compare curve labelled with squares (slope=1.15over 48 hr) vs curve labelled with dots (0.66 over 48 hr) and curvelabelled with up-side-down triangles (slope=0.94 over 74 hours) vs curvelabelled with triangles (0.79 over 74 hr). FIG. 22D. In vivotumorigenesis assay shows that TWIST1-expressing cells give rise towidespread disseminated tumours, especially lining the wall of theperitoneal cavity. In contrast, sh492-expressing cells do not colonizethe peritoneal wall. Arrows indicate carcinomatosis in mice engraftedwith Ov8GFP-TWIST1 cells. All error bars represent standard error of themean.

FIGS. 23A to 23C. RNA sequencing reveals differential expression ofGAS6, L1CAM, and HMGA2. FIG. 23A. RNA sequencing showed approximately2-fold increases in GAS6 and L1CAM and a 2-fold decrease in HMGA2 mRNAwhen TWIST1 is overexpressed. FPKM, fragments per kilobase per millionreads. FIG. 23B. Western blot confirms differential expression of L1CAMand HMGA2 found by RNA-seq. Blots cropped for clarity; full blots areshown in FIG. 30. FIG. 23C. No western blot was possible for GAS6, asthe protein is secreted, but qRT-PCR shows on average a 50% decrease inGAS6 mRNA level upon TWIST knockdown. p=0.31, although a clear trend ispresent. Error bars represent standard error of the mean.

FIGS. 24A to 24F. Knockdown of GAS6 or L1CAM reverses drug resistance.FIGS. 24A-24C. Validation of siRNAs targeting genes of interest. qRT-PCRconfirms knockdown of L1CAM (FIG. 24A) (46%) and GAS6 (FIG. 24B) (90%)in Ov8GFP-TWIST1 cells treated with corresponding siRNAs, and 91%knockdown of HMGA2 (FIG. 24C) in Ov8GFP-sh492 cells treated with HMGA2siRNAs. FIG. 24D. Western blot confirms knockdown of L1CAM and HMGA2 atthe protein level (normalized results from three independentexperiments, p=0.0276 for HMGA2, p=0.0042 for L1CAM). FIG. 24E. SRBassay demonstrates that knockdown of HMGA2 in Ov8GFP-sh492 cells is notsufficient to confer an increased resistance to cisplatin. FIG. 24F.Knockdown of either GAS6 or L1CAM in Ov8GFP-TWIST1 cells sensitizes thisline to cisplatin, compared to treatment with non-targeting siRNA. Upperasterisks, siGAS6 (1 μM, p=0.0108, 3 μM p=0.00077, 9 μM p=0.054); lower,siL1CAM (1 μM, p=0.00077, 3 μM p<0.0001, 9 μM p=0.0064). qPCR error barsrepresent minimum and maximum values calculated by the StepOne softwareanalysis. SRB error bars represent standard error of the mean.

FIGS. 25A to 25D. TWIST1, GAS6, and L1CAM expression lead toupregulation of Akt signalling following cisplatin treatment. FIG. 25A.Quantification of western blot data shows that over the course of 24 hr,5 μM cisplatin treatment leads to increased levels of Akt inOv8GFP-TWIST1 cells, but not in Ov8GFP-sh492 cells. Knockdown of GAS6 orL1CAM in Ov8GFP-TWIST1 cells partially abrogates the increase in Akt.NT, not treated with cisplatin. FIG. 25B. Western blot also reveals anincrease in activation of Akt via phosphorylation at Ser 473 over 24 hrof 5 μM cisplatin in TWIST1 expressing cells. The opposite is true inOv8GFP-sh492 cells, in which Akt activity is reduced over the same timeperiod. Knockdown of GAS6 in Ov8GFP-TWIST1 cells maintains a constantpAkt/Akt ratio, while L1CAM knockdown partially prevents Akt activation.FIG. 25C. Ov8GFP-TWIST1 cells further increase their TWIST1 expressionup to 2.3 fold over 24 hr of exposure to 5 μM cisplatin (p=0.0827),whereas Ov8GFP-sh492 cells show no increase. FIG. 25D. Treatment ofOv8GFP-TWIST1 cells with the PI3K inhibitor LY294002 to prevent Aktactivation sensitized cells to cisplatin, compared to DMSO only control,supporting the assertion that Akt signalling is central to TWIST1-drivencisplatin resistance. p<0.0001 for both concentrations. Error barsrepresent standard error of the mean.

FIG. 26. Schematic representation of our proposed model. TWISTupregulates expression of GAS6 and L1CAM. TWIST1, GAS6, and L1CAMfacilitate phosphorylation and activation of Akt, leading to increasedproliferation. This in turn results in cisplatin resistance and greatertumour cell engraftment in vivo.

FIGS. 27A to 27B. Cells transfected with empty pCI-Neo vector exhibitintermediate phenotype. FIG. 27A. Quantification of representativewestern blot data demonstrating that TWIST1 expression is reduced 2-foldby sh492 and increased approximately 3.5-fold by expression of a secondcopy of the gene. FIG. 27B. Transfection of cells with empty pCI-Neovector does not substantially affect TWIST1 expression levels.Representative western data shown.

FIG. 28. Representative images of Ov8GFP-TWIST and Ov8GFP-sh492 cellsacquired three days post treatment with the indicated doses ofcisplatin. Images such as these were analyzed to determine confluence,which is graphed in FIG. 22C.

FIG. 29. Representative images of Ov8GFP-sh492 and Ov8GFP-TWIST1 tumoursfrom mice, immunostained for cell surface L1CAM. Ov8GFP-sh492 tumourswere heterogeneous, with different tumour masses displaying no staining(top), light staining (middle) or dark staining. TWIST1 tumours werestained more uniformly and were typically dark with some lighter areas.

FIG. 30. Example of raw data for western blot graphs shown in FIGS.25A-25C. Increases in total and activated Akt can be seen for TWIST siQ,and is abrogated in other conditions tested. Absence of L1CAM and TWIST1can be observed in siL1CAM and sh492 lanes, respectively. All samplesnormalized to actin (bottom).

FIGS. 31A to 31B. NSG female mice were injected with OVCAR8-ip cellsintraperitoneally, grown for two weeks, then treated IP with siRNAagainst Twist 1 or control (siQ), and one week later treated withcisplatin IP. Results indicate significant reduction in weights ofmetastases when siRNA-TWIST was combined with cisplatin. FIG. 31A showedthe tumor weight of each experimental group. FIG. 31B showed the tumorimaging of Group B and Group F.

FIG. 32. Ovcar8-GFP-ffluc cells were stably transfected with CMV-drivenshRNA against TWIST1 (sh492) or CMV-driven TWIST1. Ovcar8 TWIST cellstreated for three days with cisplatin showed markedly higher survivalcompared to Ovcar8 sh492 cells, demonstrating a role for TWIST1 incisplatin resistance.

FIG. 33. Ovcar8-GFP-ffluc cells stably transfected with sh492 or TWISTwere injected IP into NSG mice. After 7 weeks, TWIST expressing cellshad given rise to carcinomatosis throughout the abdomen, whereas sh492cells did not. sh492 cells also gave rise to smaller masses in theovary.

FIG. 34. Human SUM1315 cells become more sensitive to doxorubicin (top)and paclitaxel (bottom) treatment in vitro upon knockdown of TWIST, inthis case by stable transfection with shRNA targeting TWIST (sh418).sh418 includes SEQ ID NO: 1 of anti-TWIST siRNA, and is used forestablishing stable cell lines using adenoviral transduction.

FIG. 35. Stable transfection of shRNA targeting TWIST led to reducedtumor volume in the 4T1 syngeneic mouse tumor in Balb/C mice (n=4). MSNtransfection of 4T1 cells demonstrates knockdown of TWIST at one weekpost transfection in vitro.

DETAILED DESCRIPTION I. Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

The use of a singular indefinite or definite article (e.g., “a,” “an,”“the,” etc.) in this disclosure and in the following claims follows thetraditional approach in patents of meaning “at least one” unless in aparticular instance it is clear from context that the term is intendedin that particular instance to mean specifically one and only one.Likewise, the term “comprising” is open ended, not excluding additionalitems, features, components, etc. References identified herein areexpressly incorporated herein by reference in their entireties unlessotherwise indicated.

The terms “comprise,” “include,” and “have,” and the derivativesthereof, are used herein interchangeably as comprehensive, open-endedterms. For example, use of “comprising,” “including,” or “having” meansthat whatever element is comprised, had, or included, is not the onlyelement encompassed by the subject of the clause that contains the verb.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and 0-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may In embodiments be conjugated to a moiety thatdoes not consist of amino acids. The terms apply to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that are recombinantlyexpressed as a single moiety.

As may be used herein, the terms “nucleic acid,” “nucleic acidmolecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acidsequence,” “nucleic acid fragment” and “polynucleotide” are usedinterchangeably and are intended to include, but are not limited to, apolymeric form of nucleotides covalently linked together that may havevarious lengths, either deoxyribonucleotides or ribonucleotides, oranalogs, derivatives or modifications thereof. Different polynucleotidesmay have different three-dimensional structures, and may perform variousfunctions, known or unknown. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the invention maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the compliment of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. The present invention includesnucleic acids sequences that are substantially identical to any of SEQID NOs: 1-21.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence with a higher affinity, e.g., under more stringentconditions, than to other nucleotide sequences (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent hybridization conditions areselected to be about 5-10° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent hybridization conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal is at least twotimes background, preferably 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley& Sons.

For specific proteins described herein (e.g., TWIST, including TWIST1and TWIST2; or proteins listed in Table 1), the named protein includesany of the protein's naturally occurring forms, or variants thatmaintain the protein transcription factor activity (e.g., within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native protein). In some embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring form.In other embodiments, the protein is the protein as identified by itsNCBI sequence reference. In other embodiments, the protein is theprotein as identified by its NCBI sequence reference or functionalfragment thereof.

For specific genes described herein (e.g., TWIST, including TWIST1,TWIST2; or genes listed in Table 1), the named gene includes any of thegene's naturally occurring forms, or variants that encode proteins thatmaintain the protein activity (e.g., within at least 50%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100% activity compared to the native protein). Insome embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or100% nucleic acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous nucleicacid portion) compared to a naturally occurring form. In otherembodiments, the gene is the gene as identified by its NCBI sequencereference. In other embodiments, the gene is 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the gene as identified by its NCBIsequence reference or functional fragment thereof.

A “TWIST1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Twist Family BHLHTranscription Factor 1 (TWIST1), homologs or variants thereof thatmaintain TWIST1 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to TWIST1). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring TWIST1 polypeptide (NCBI referencenumber: NP_000465.1 or Gene ID: GI:4507741). In embodiments, the TWIST1gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number Gene ID: 68160957(NM_000474.3) or a variant having substantial identity thereto.

A “TWIST2 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Twist Family BHLHTranscription Factor 2 (TWIST2), homologs or variants thereof thatmaintain TWIST2 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to TWIST2). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring TWIST2 polypeptide (NCBI referencenumber: NP_001258822.1 or Gene ID:429325228). In embodiments, the TWIST2gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number Gene ID: 618468605(NM_001271893.3) or a variant having substantial identity thereto.

A “HCLS1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding hematopoietic lineagecell-specific protein 1 (HCLS1), homologs or variants thereof thatmaintain HCLS1 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to HCLS1). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring HCLS1 polypeptide (NCBI referencenumber: NP_001278970 or NP_005326.2). In embodiments, the HCLS1 gene is80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleicacid identified by the NCBI reference number NM_001292041 or NM_005335.5or a variant having substantial identity thereto.

A “ESM1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding endothelial cell specificmolecule 1 (ESM1), homologs or variants thereof that maintain ESM1protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to ESM1). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring ESM1 polypeptide (NCBI reference number:NP_001129076 or NP_008967). In embodiments, the ESM1 gene is 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_001135604 or NM_007036 or avariant having substantial identity thereto.

A “GGT1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding gamma-glutamyltransferase1 (GGT1), homologs or variants thereof that maintain GGT1 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to GGT1). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringGGT1 polypeptide (NCBI reference number: NP_001275762). In embodiments,the GGT1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid identified by the NCBI reference numberNM_001288833 or a variant having substantial identity thereto.

A “TIE1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding tyrosine kinase withimmunoglobulin like and EGF like domains 1 (TIE1), homologs or variantsthereof that maintain TIE1 protein activity (e.g. within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to TIE1). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring TIE1 polypeptide (NCBI referencenumber: NP_001240286 or NP_005415.1). In embodiments, the TIE1 gene is80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleicacid identified by the NCBI reference number NM_001253357 or NM_005424.4or a variant having substantial identity thereto.

A “CCL20 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding C-C motif chemokineligand 20 (CCL20), homologs or variants thereof that maintain CCL20protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to CCL20). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring CCL20 polypeptide (NCBI reference number:NP_001123518 or NP:_004582). In embodiments, the CCL20 gene is 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_001130046 or NM_004591 or avariant having substantial identity thereto.

An “ABCA1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding ATP Binding CassetteSubfamily A Member 1 (ABCA1), homologs or variants thereof that maintainABCA1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to ABCA1). In embodiments,variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring ABCA1 polypeptide (NCBI reference number:NP_005493). In embodiments, the ABCA1 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_005502 or a variant having substantial identitythereto.

A “SP4 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Sp4 transcription factor,homologs or variants thereof that maintain SP4 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to SP4). In embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring SP4polypeptide (NCBI reference number: NP_001313471, NP_001313472, orNP_003103). In embodiments, the SP4 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_001326542, NM_001326543, NM_003112 or a varianthaving substantial identity thereto.

An “ARHGDIB gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding Rho GDP-dissociationinhibitor 2 (ARHGDIB), homologs or variants thereof that maintainARHGDIB protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to ARHGDIB). In embodiments,variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring ARHGDIB polypeptide (NCBI reference number:NP_001166). In embodiments, the ARHGDIB gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_001175.6 or a variant having substantialidentity thereto.

A “LSAMP gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding limbic system-associatedmembrane protein (LSAMP), homologs or variants thereof that maintainLSAMP protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to LSAMP). In embodiments,variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring LSAMP polypeptide (NCBI reference number:NP_001305844 or NP_002329). In embodiments, the LSAMP gene is 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_001318915, NM_002338 or avariant having substantial identity thereto.

An “EDN1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Endothelin 1 (EDN1),homologs or variants thereof that maintain EDN1 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to EDN1). In embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring EDN1polypeptide (NCBI reference number. NP_001161791 or NP_001946). Inembodiments, the EDN1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% identical to the nucleic acid identified by the NCBI referencenumber NM_0011683198915, NM_001955 or a variant having substantialidentity thereto.

An “IL1A gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding interleukin 1 alpha(IL1A), homologs or variants thereof that maintain IL1A protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to IL1A). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring IL1Apolypeptide (NCBI reference number: NP_000566). In embodiments, the IL1Agene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NM_000575 or avariant having substantial identity thereto.

A “CTPS2 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding CTP synthase 2 (CTPS2),homologs or variants thereof that maintain CTPS2 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CTPS2). In embodiments, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring CTPS2polypeptide (NCBI reference number: NP_001137474, NP_062831, orNP_787055). In embodiments, the CTPS2 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_001144002, NM_019857, NM_175859 or a varianthaving substantial identity thereto.

A “SERPINB2 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding serpin family B member2 (SERPINB2), homologs or variants thereof that maintain SERPINB2protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to SERPINB2). In embodiments,variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring SERPINB2 polypeptide (NCBI reference number:NP_001137290, or NP_002566). In embodiments, the SERPINB2 gene is 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_001143818, NM_002575, or avariant having substantial identity thereto.

A “HMGA2 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding high mobility groupAT-hook 2 (HMGA2), homologs or variants thereof that maintain HMGA2protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to HMGA2). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring HMGA2 polypeptide (NCBI reference number:NP_001287847, NP_001287848, NP_001317119, NP_003474, or NP_003475). Inembodiments, the HMGA2 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% identical to the nucleic acid identified by the NCBI referencenumber NM_001300918, NM_001300919, NM_001330190, NM_003483, NM_003484,or a variant having substantial identity thereto.

A “LOC643201 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the centrosomal protein 192 kDapseudogene (LOC643201), homologs or variants thereof that maintain itsprotein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to LOC643201). In embodiments,variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring LOC643201 polypeptide. In embodiments, the LOC643201gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NR_036494 or avariant having substantial identity thereto.

A “LPHN2 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding adhesion Gprotein-coupled receptor L2 (LPHN2), homologs or variants thereof thatmaintain LPHN2 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to LPHN2). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring LPHN2 polypeptide (NCBI referencenumber: NP_001284633). In embodiments, the LPHN2 gene is 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identifiedby the NCBI reference number NM_001297704 or a variant havingsubstantial identity thereto.

A “CALB1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding calbindin 1 (CALB1),homologs or variants thereof that maintain CALB1 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CALB1). In embodiments, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring CALB1polypeptide (NCBI reference number: NP_004920). In embodiments, theCALB1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the nucleic acid identified by the NCBI reference number NM_004929 ora variant having substantial identity thereto.

A “FAM129A gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding family with sequencesimilarity 129 member A (FAM129A), homologs or variants thereof thatmaintain FAM129A protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to FAM129A). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring FAM129A polypeptide (NCBI referencenumber: NP_443198). In embodiments, the FAM129A gene is 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identifiedby the NCBI reference number NM_052966 or a variant having substantialidentity thereto.

A “DPYSL3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding dihydropyrimidinase like3 (DPYSL3), homologs or variants thereof that maintain DPYSL3 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to DPYSL3). In embodiments, variants have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring DPYSL3 polypeptide (NCBI reference number: NP_001184223 orNP_001378). In embodiments, the DPYSL3 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_001197294, NM_001387 or a variant havingsubstantial identity thereto.

A “GAS6 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding growth arrest specific 6(GAS6), homologs or variants thereof that maintain GAS6 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to GAS6). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring GAS6polypeptide (NCBI reference number: NP_000811). In embodiments, the GAS6gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NM_000820 or avariant having substantial identity thereto.

A “CHN1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding chimerin 1 (CHN1),homologs or variants thereof that maintain CHN1 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CHN1). In embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring CHN1polypeptide (NCBI reference number: NP_001020372, NP_001193531 orNP_001813). In embodiments, the CHN1 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_001025201, NM_001206602, NM_001822 or a varianthaving substantial identity thereto.

A “GREM1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding gremlin 1, DAN family BMPantagonist (GREM1), homologs or variants thereof that maintain GREM1protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to GREM1). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring GREM1 polypeptide (NCBI reference number:NP_001178251, NP_001178252 or NP_037504). In embodiments, the GREM1 geneis 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NM_001191322,NM_001191323, NM_013372 or a variant having substantial identitythereto.

A “SHISA9 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding shisa family member 9(SHISA9), homologs or variants thereof that maintain SHISA9 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to SHISA9). In embodiments, variants have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring SHISA9 polypeptide (NCBI reference number: NP_001138676, orNP_001138677). In embodiments, the SHISA9 gene is 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified bythe NCBI reference number NM_001145204, NM_001145205 or a variant havingsubstantial identity thereto.

An “EPHB1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding Ephrin type-B receptor 1(EPHB1), homologs or variants thereof that maintain EPHB1 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to EPHB1). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringEPHB1 polypeptide (NCBI reference number: NM_004441). In embodiments,the EPHB1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid identified by the NCBI reference numberNM_004441 or a variant having substantial identity thereto.

A “RHOBTB1 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding Rho-related BTBdomain-containing protein 1 (RHOBTB1), homologs or variants thereof thatmaintain RHOBTB1 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to RHOBTB1). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring RHOBTB1 polypeptide (NCBI referencenumber: NP_001229288). In embodiments, the RHOBTB1 gene is 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_001242359 or a variant havingsubstantial identity thereto.

A “L1CAM gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding L1 cell adhesion molecule(L1CAM), homologs or variants thereof that maintain L1CAM proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to L1CAM). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringL1CAM polypeptide (NCBI reference number: NP_000416.1, NP_001137435.1,NP_001265045.1 or NP_076493.1). In embodiments, the L1CAM gene is 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_000425.4, NM_001143963.2,NM_001278116.1, NM_024003.3 or a variant having substantial identitythereto.

A “PCSK9 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding proprotein convertasesubtilisin/kexin type 9 (PCSK9), homologs or variants thereof thatmaintain PCSK9 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to PCSK9). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring PCSK9 polypeptide (NCBI referencenumber: NP_777596). In embodiments, the PCSK9 gene is 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid identifiedby the NCBI reference number NM_174936 or a variant having substantialidentity thereto.

An “OAS3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding 2′-5′-oligoadenylatesynthase 3 (OAS3), homologs or variants thereof that maintain OAS3protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to OAS3). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring OAS3 polypeptide (NCBI reference number: NP_006178).In embodiments, the OAS3 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the nucleic acid identified by the NCBI referencenumber NM_006187 or a variant having substantial identity thereto.

A “LOX gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding protein-lysine 6-oxidase(LOX), homologs or variants thereof that maintain LOX protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to LOX). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring LOXpolypeptide (NCBI reference number: NP_002308). In embodiments, the LOXgene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NM_002317 or avariant having substantial identity thereto.

A “DOK7 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding protein docking protein 7(DOK7), homologs or variants thereof that maintain DOK7 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to DOK7). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring DOK7polypeptide (NCBI reference number: NP_001158145). In embodiments, theDOK7 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical tothe nucleic acid identified by the NCBI reference number NM_001164673 ora variant having substantial identity thereto.

An “ID3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding inhibitor of DNA binding3 (ID3), homologs or variants thereof that maintain ID3 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to ID3). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring ID3polypeptide (NCBI reference number: NP_002158). In embodiments, the ID3gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NM_002167 or avariant having substantial identity thereto.

A “LEPREL1 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding prolyl 3-hydroxylase 2(LEPREL1), homologs or variants thereof that maintain LEPREL1 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to LEPREL1). In embodiments, variants have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring LEPREL1 polypeptide (NCBI reference number: NP_001127890). Inembodiments, the LEPREL1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the nucleic acid identified by the NCBI referencenumber NM_001134418 or a variant having substantial identity thereto.

A “CAPN6 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding calpain-6 (CAPN6),homologs or variants thereof that maintain CAPN6 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to CAPN6). In embodiments, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring CAPN6polypeptide (NCBI reference number: NP_055104). In embodiments, theCAPN6 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the nucleic acid identified by the NCBI reference number NM_014289 ora variant having substantial identity thereto.

A “FN1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding fibronectin 1 (FN1),homologs or variants thereof that maintain FN1 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to FN1). In embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring FN1polypeptide (NCBI reference number: NP_001293058). In embodiments, theFN1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical tothe nucleic acid identified by the NCBI reference number NM_001306129 ora variant having substantial identity thereto.

A “COL12A1 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding collagen alpha-1(XII)chain (COL12A1), homologs or variants thereof that maintain COL12A1protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to COL12A1). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring COL12A1 polypeptide (NCBI reference number:NP_004361). In embodiments, the COL12A1 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_004370 or a variant having substantial identitythereto.

An “AMIGO2 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding amphoterin-inducedprotein 2 (AMIGO2), homologs or variants thereof that maintain AMIGO2protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to AMIGO2). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring AMIGO2 polypeptide (NCBI reference number:NP_001137140). In embodiments, the AMIGO2 gene is 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to the nucleic acid identified bythe NCBI reference number NM_001143668 or a variant having substantialidentity thereto.

A “GALNT3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding polypeptideN-acetylgalactosaminyltransferase 3 (GALNT3), homologs or variantsthereof that maintain GALNT3 protein activity (e.g. within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to GALNT3).In embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity across the whole sequence or a portionof the sequence (e.g. a 50, 100, 150 or 200 continuous amino acidportion) compared to a naturally occurring GALNT3 polypeptide (NCBIreference number: NP_004473). In embodiments, the GALNT3 gene is 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_004482 or a variant havingsubstantial identity thereto.

A “COL4A4 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding collagen alpha-4(IV)chain (COL4A4), homologs or variants thereof that maintain COL4A4protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to COL4A4). In embodiments, variantshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring COL4A4 polypeptide (NCBI reference number:NP_000083). In embodiments, the COL4A4 gene is 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the nucleic acid identified by theNCBI reference number NM_000092 or a variant having substantial identitythereto.

A “HOXA3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding homeobox protein Hox-A3(HOXA3), homologs or variants thereof that maintain HOXA3 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to HOXA3). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringHOXA3 polypeptide (NCBI reference number: NP_109377). In embodiments,the HOXA3 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid identified by the NCBI reference numberNM_030661 or a variant having substantial identity thereto.

An “ATOH8 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding protein atonal homolog 8(ATOH8), homologs or variants thereof that maintain ATOH8 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to ATOH8). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringATOH8 polypeptide (NCBI reference number: NP_116216). In embodiments,the ATOH8 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid identified by the NCBI reference numberNM_032827 or a variant having substantial identity thereto.

A “GDF6 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding growth/differentiationfactor 6 (GDF6), homologs or variants thereof that maintain GDF6 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to GDF6). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringGDF6 polypeptide (NCBI reference number: NP_001001557). In embodiments,the GDF6 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid identified by the NCBI reference numberNM_001001557 or a variant having substantial identity thereto.

A “PXDNL gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding peroxidasin-like protein(PXDNL), homologs or variants thereof that maintain PXDNL proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to PXDNL). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurringPXDNL polypeptide (NCBI reference number: NP_653252). In embodiments,the PXDNL gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the nucleic acid identified by the NCBI reference numberNM_144651 or a variant having substantial identity thereto.

A “BDKRB1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding B1 bradykinin receptor 1(BDKRB1), homologs or variants thereof that maintain BDKRB1 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to BDKRB1). In embodiments, variants have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring BDKRB1 polypeptide (NCBI reference number: NP_000701). Inembodiments, the BDKRB1 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the nucleic acid identified by the NCBI referencenumber NM_000710 or a variant having substantial identity thereto.

A “LINC00452 gene” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene long intergenic non-proteincoding RNA 452 (LINC00452), homologs or variants thereof that maintainits protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to wild type protein). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring LINC00452 polypeptide (NCBI referencenumber: NP_001265603). In embodiments, the LINC00452 gene is 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acididentified by the NCBI reference number NM_001278674 or a variant havingsubstantial identity thereto.

A “VIP gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding vasoactive intestinalpeptide (VIP), homologs or variants thereof that maintain VIP proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to VIP). In embodiments, variants have at least90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity acrossthe whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or200 continuous amino acid portion) compared to a naturally occurring VIPpolypeptide (NCBI reference number: NP_003372 or NP_919416.1). Inembodiments, the VIP gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% identical to the nucleic acid identified by the NCBI referencenumber NM_003381, NM_194435.2, or a variant having substantial identitythereto.

A “DPT gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding dermatopontin (DPT),homologs or variants thereof that maintain DPT protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to DPT). In embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring DPTpolypeptide (NCBI reference number: NP_001928). In embodiments, the DPTgene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid identified by the NCBI reference number NM_001937 or avariant having substantial identity thereto.

A “KCNA10 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding B1 bradykinin receptor 1(KCNA10), homologs or variants thereof that maintain KCNA10 proteinactivity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or100% activity compared to KCNA10). In embodiments, variants have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring KCNA10 polypeptide (NCBI reference number: NP_005540). Inembodiments, the KCNA10 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the nucleic acid identified by the NCBI referencenumber NM_005549 or a variant having substantial identity thereto.

A “microRNA,” “microRNA nucleic acid sequence,” “miR,” “miRNA” as usedherein, refers to a nucleic acid that functions in RNA silencing andpost-transcriptional regulation of gene expression. The term includesall forms of a miRNA, such as the pri-, pre-, and mature forms of themiRNA. In embodiments, microRNAs (miRNAs) are short (20-24 nt)non-coding RNAs that are involved in post-transcriptional regulation ofgene expression in multicellular organisms by affecting both thestability and translation of mRNAs. miRNAs are transcribed by RNApolymerase II as part of capped and polyadenylated primary transcripts(pri-miRNAs) that can be either protein-coding or non-coding. Theprimary transcript is cleaved by the Drosha ribonuclease III enzyme toproduce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA),which is further cleaved by the cytoplasmic Dicer ribonuclease togenerate the mature miRNA and antisense miRNA star (miRNA*) products.The mature miRNA is incorporated into a RNA-induced silencing complex(RISC), which recognizes target mRNAs through imperfect base pairingwith the miRNA and most commonly results in translational inhibition ordestabilization of the target mRNA. In embodiments, a miRNA nucleic acidsequence described herein is about 10 to 80 nucleotides (e.g., 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 nucleotides)in length. In embodiments, a miRNA nucleic acid sequence describedherein is about 15 to 50 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides) in length. Inembodiments, a miRNA nucleic acid sequence described herein is about 18to 25 nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25 nucleotides) inlength.

As used herein, the term “miR484” or “miR484 nucleic acid sequence”includes all forms of miR484 including the pri-, pre-, and mature formsof miR484, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR484). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR484 is the miRNA as identified by NCBI ReferenceSequence: NR_030159.

As used herein, the term “miR1909” or “miR1909 nucleic acid sequence”includes all forms of miR1909 including the pri-, pre-, and mature formsof miR1909, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR1909). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR1909 is the miRNA as identified by NCBI ReferenceSequence: NR_031730.

As used herein, the term “miR5193” or “miR5193 nucleic acid sequence”includes all forms of miR5193 including the pri-, pre-, and mature formsof miR5193, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miRS193). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR5193 is the miRNA as identified by NCBI ReferenceSequence: NR_049825.

As used herein, the term “miR4324” or “miR4324 nucleic acid sequence”includes all forms of miR4324 including the pri-, pre-, and mature formsof miR4324, as well as variants, homologues, modifications, andderivatives thereof (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%,98%, 99% or 100% activity compared to the native miR4324). Inembodiments, the variants or homologues or derivatives have at least50%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity across the whole sequence or a portion of the sequence(e.g. a 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, or 80continuous nucleotides portion) compared to a naturally occurring form.In embodiments, the miR4324 is the miRNA as identified by NCBI ReferenceSequence: NR_036209.

An “siRNA” refers to a nucleic acid that forms a double stranded RNA,which double stranded RNA has the ability to reduce or inhibitexpression of a gene or target gene when the siRNA is present (e.g.expressed) in the same cell as the gene or target gene. The siRNA istypically about 5 to about 100 nucleotides in length, more typicallyabout 10 to about 50 nucleotides in length, more typically about 15 toabout 30 nucleotides in length, most typically about 20-30 basenucleotides, or about 20-25 or about 24-29 nucleotides in length, e.g.,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.siRNA molecules and methods of generating them are described in, e.g.,Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411,494-498; WO 00/44895; WO 01/36646; WO 99/32619; WO 00/01846; WO01/29058; WO 99/07409; and WO 00/44914. A DNA molecule that transcribesdsRNA or siRNA (for instance, as a hairpin duplex) also provides RNAi.DNA molecules for transcribing dsRNA are disclosed in U.S. Pat. No.6,573,099, and in U.S. Patent Application Publication Nos. 2002/0160393and 2003/0027783, and Tuschl and Borkhardt, Molecular Interventions,2:158 (2002).

Of the double stranded RNA of an siRNA, the strand that is at leastpartially complementary to at least a portion of a specific targetnucleic acid (e.g. a target nucleic acid sequence), such as an mRNAmolecule (e.g. a target mRNA molecule), is called the antisense (orguide strand; and the other strand is called sense (or passengerstrand). The passenger strand is degraded and the guide strand isincorporated into the RNA-induced silencing complex (RISC).

The siRNA can be administered directly or siRNA expression vectors canbe used to induce RNAi that have different design criteria. A vector canhave inserted two inverted repeats separated by a short spacer sequenceand ending with a string of T's which serve to terminate transcription.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a linear orcircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g., nonepisomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions. Additionally, some viralvectors are capable of targeting a particular cells type eitherspecifically or non-specifically. Replication-incompetent viral vectorsor replication-defective viral vectors refer to viral vectors that arecapable of infecting their target cells and delivering their viralpayload, but then fail to continue the typical lytic pathway that leadsto cell lysis and death.

The compositions described herein can be purified. Purified compositionsare at least about 60% by weight (dry weight) the compound of interest.Preferably, the preparation is at least about 75%, more preferably atleast about 90%, and most preferably at least about 99% or higher byweight the compound of interest. Purity is measured by any appropriatestandard method, for example, by High-performance liquid chromatography,polyacrylamide gel electrophoresis.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance that results in a detectably lowerexpression or activity level as compared to a control. The inhibitedexpression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or less than that in a control. In certain instances, the inhibitionis 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more incomparison to a control. An “inhibitor” is a siRNA, (e.g., shRNA, miRNA,snoRNA), compound or small molecule that inhibits cellular function(e.g., replication) e.g., by binding, partially or totally blockingstimulation, decrease, prevent, or delay activation, or inactivate,desensitize, or down-regulate signal transduction, gene expression orenzymatic activity necessary for protein activity. A “TWIST inhibitor”refers to a substance that results in a detectably lower expression ofTWIST (TWIST1, TWIST2 or both) genes or TWIST (TWIST1, TWIST2 or both)proteins or lower activity level of TWIST (TWIST1, TWIST2 or both)proteins as compared to those levels without such substance. In someembodiments, a TWIST inhibitor is an inhibitor for both TWIST1 andTWIST2. In some embodiments, a TWIST inhibitor is an anti-TWIST siRNA.In some embodiments, a TWIST inhibitor is a composition (e.g., ananti-TWIST siRNA bound to a nanoparticle) described herein. In someembodiments, a TWIST inhibitor is a pharmaceutical composition describedherein.

“TWIST signaling” or “TWIST signaling pathway” used herein refers to theintracellular signaling pathway activated when TWIST (TWIST1 or TWIST2or both TWIST1 and TWIST2) binds to DNA and initiates itstranscriptional activity. Activation of TWIST initiates functioning ofmany downstream factors and signaling pathways. “TWIST signaling gene”refers to a gene in the TWIST signaling. In embodiments, TWIST signalinggenes include TWIST1, TWIST2, genes listed in Table 1 and an Akt (orAkt/PI3K) signaling gene. “TWIST signaling protein” refers to a proteinin the TWIST signaling protein. In embodiments, TWIST signaling proteinsinclude TWIST1, TWIST2, proteins listed in Table 1 and an Akt (orAkt/PI3K) signaling protein.

“Akt (or Akt/PI3K) signaling” or “Akt (or Akt/PI3K) signaling pathway”used herein refers to intracellular signaling pathway activated whengrowth factor causes activation of a cell surface receptor andphosphorylation of PI3K. Activated PI3K then phosphorylates lipids onthe plasma membrane, forming second messenger phosphatidylinositol(3,4,5)-trisphosphate (PIP3). Akt, a serine/threonine kinase, isrecruited to the membrane by interaction with these phosphoinositidedocking sites, so that it can be fully activated. Activated Akt mediatesdownstream responses, including cell survival, growth, proliferation,cell migration and angiogenesis, by phosphorylating a range ofintracellular proteins. “Akt/PI3K signaling gene” refers to a gene inthe Akt/PI3K signaling. In embodiments, Akt/PI3K signaling genes includeAkt (e.g., Akt1, Akt2, or Akt3) and PI3K genes. “Akt/PI3K signalingprotein” refers to a protein in the Akt/PI3K signaling. In embodiments,Akt/PI3K signaling proteins include Akt (e.g., Akt1, Akt2, or Akt3) andPI3K proteins.

An “Akt1 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding RAC-alphaserine/threonine-protein kinase (Akt1), homologs or variants thereofthat maintain Akt1protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to Akt1). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring Akt1 polypeptide (NCBI referencenumber: NP_001014432.1 or NP_005154.2). In embodiments, the Akt1 gene is80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleicacid identified by the NCBI reference number NM_001014432.1, NM_005163.2or a variant having substantial identity thereto.

An “Akt1” as referred to herein includes any of the recombinant ornaturally-occurring forms of RAC-alpha serine/threonine-protein kinase(Akt1), homologs or variants thereof that maintain Akt1 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to Akt1). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring Akt1protein. In embodiments, variants have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to amino acid sequence identified by NCBIreference number: NP_001014432.1 or NP_005154.2.

An “Akt2 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding RAC-betaserine/threonine-protein kinase (Akt2), homologs or variants thereofthat maintain Akt2 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to Akt2). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring Akt2 polypeptide (NCBI referencenumber: NP_001229956.1 or NP_001229957.1, NP_001317440.1, NP_001617.1).In embodiments, the Akt2 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the nucleic acid identified by the NCBI referencenumber NM_001243027.2, NM_001243028.2, NM_001330511.1, NM_001626.5 or avariant having substantial identity thereto.

An “Akt2” as referred to herein includes any of the recombinant ornaturally-occurring forms of RAC-beta serine/threonine-protein kinase(Akt2), homologs or variants thereof that maintain Akt2 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to Akt2). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring Akt2protein. In embodiments, variants have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to amino acid sequence identified by NCBIreference number: NM_001243027.2, NM_001243028.2, NM_001330511.1, orNM_001626.5.

An “Akt3 gene” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding RAC-gammaserine/threonine-protein kinase (Akt3), homologs or variants thereofthat maintain Akt2 protein activity (e.g. within at least 50%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% activity compared to Akt3). Inembodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring Akt3 polypeptide (NCBI referencenumber: NP_001193658.1, NP_005456.1, NP_859029.1, or NP_001617.1). Inembodiments, the Akt3 gene is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% identical to the nucleic acid identified by the NCBI referencenumber NM_001206729.1, NM_005465.4, NM_181690.2 or a variant havingsubstantial identity thereto.

An “Akt3” as referred to herein includes any of the recombinant ornaturally-occurring forms of RAC-gamma serine/threonine-protein kinase(Akt3), homologs or variants thereof that maintain Akt3 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to Akt3). In embodiments, variants have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring Akt3protein. In embodiments, variants have at least 90%, 95%, 96%, 97%, 98%,99% or 100% amino acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 50, 100, 150 or 200 continuous aminoacid portion) compared to amino acid sequence identified by NCBIreference number: NP_001193658.1, NP_005456.1, NP_859029.1, orNP_001617.1.

A “PI3K” as referred to herein includes any of the recombinant ornaturally-occurring forms of phosphatidylinositide 3-kinases (PI3K),homologs or variants thereof that maintain Akt3 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to PI3K). In embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring PI3Kprotein.

In embodiments, PI3K is class I PI3K. Class I PI3Ks are responsible forthe production of phosphatidylinositol 3-phosphate (PI(3)P),phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), andphosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3). Class I PI3Kare heterodimeric molecules composed of a regulatory and a catalyticsubunit; they are further divided between IA and IB subsets on sequencesimilarity. Class IA PI3K is composed of a heterodimer between a p110catalytic subunit and a p85 regulatory subunit. There are five variantsof the p85 regulatory subunit, designated p85α, p55α, p50α, p85β, andp55γ. There are also three variants of the p110 catalytic subunitdesignated p110α, β, or δ catalytic subunit. The first three regulatorysubunits are all splice variants of the same gene (Pik3r1), the othertwo being expressed by other genes (Pik3r2 and Pik3r3, p85β, and p557,respectively). The most highly expressed regulatory subunit is p85a; allthree catalytic subunits are expressed by separate genes (Pik3ca,Pik3cb, and Pik3cd for p110α, p110β, and p110δ, respectively). The firsttwo p110 isoforms (α and β) are expressed in all cells, but p110δ isexpressed primarily in leukocytes, and it has been suggested that itevolved in parallel with the adaptive immune system. The regulatory p101and catalytic p110γ subunits comprise the class IB PI3Ks and are encodedby a single gene each. The p85 subunits contain SH2 and SH3 domains. TheSH2 domains bind preferentially to phosphorylated tyrosine residues inthe amino acid sequence context Y-X-X-M.

In embodiments, PI3K is class II and III PI3K. Class II and III PI3K aredifferentiated from the Class I by their structure and function. Thedistinct feature of Class II PI3Ks is the C-terminal C2 domain. Thisdomain lacks critical Asp residues to coordinate binding of Ca2+, whichsuggests class II PI3Ks bind lipids in a Ca2+-independent manner. ClassII comprises three catalytic isoforms (C2α, C2β, and C2γ), but, unlikeClasses I and III, no regulatory proteins. Class II catalyzes theproduction of PI(3)P from PI and PI(3,4)P2 from PIP; however, little isknown about their role in immune cells. C2α and C2β are expressedthrough the body, but expression of C2γ is limited to hepatocytes. ClassIII produces only PI(3)P from PI but are more similar to Class I instructure, as they exist as heterodimers of a catalytic (Vps34) and aregulatory (Vps15/p150) subunits. Class III seems to be primarilyinvolved in the trafficking of proteins and vesicles.

Exemplary PI3K genes/proteins include:

group gene protein aliases EC number class 1 catalytic PIK3CA PI3K,catalytic, alpha polypeptide p110-α 2.7.1.153 PIK3CB PI3K, catalytic,beta polypeptide p110-β PIK3CG PI3K, catalytic, gamma polypeptide p110-γPIK3CD PI3K, catalytic, delta polypeptide p110-δ class 1 regulatoryPIK3R1 PI3K, regulatory subunit 1 (alpha) p85-α N/A PIK3R2 PI3K,regulatory subunit 2 (beta) p85-β PIK3R3 PI3K, regulatory subunit 3(gamma) p55-γ PIK3R4 PI3K, regulatory subunit 4 p150 PIK3R5 PI3K,regulatory subunit 5 p101 PIK3R6 PI3K, regulatory subunit 6 p87 class 2PIK3C2A PI3K, class 2, alpha polypeptide PI3K-C2α 2.7.1.154 PIK3C2BPI3K, class 2, beta polypeptide PI3K-C2β PIK3C2G PI3K, class 2, gammapolypeptide PI3K-C2γ class 3 PIK3C3 PI3K, class 3 Vps34 2.7.1.137

A “TWIST signaling inhibitor” refers to a substance that results in adetectably lower expression and/or activity of one or more TWISTsignaling genes/proteins as compared to those levels without suchsubstance. In embodiments, a TWIST signaling inhibitor results in adetectably lower gene expression of one or more TWIST signaling genes.In embodiments, a TWIST signaling inhibitor results in a detectablylower protein expression of one or more TWIST signaling proteins. Inembodiments, a TWIST signaling inhibitor results in lower activity levelof one or more TWIST signaling proteins compared to those levels withoutsuch inhibitor. In some embodiments, a TWIST signaling inhibitor is asiRNA inhibitor. In some embodiments, a TWIST signaling inhibitor is asmall molecule inhibitor. In embodiments, a TWIST signaling inhibitor isa TWIST inhibitor, an inhibitor of genes/proteins listed in Table 1and/or an Akt signaling genes/proteins. In embodiments, a TWISTsignaling inhibitor is a TWIST inhibitor, an inhibitor of GAS6, L1CAM,PI3K or protein kinase B (also known as Akt). In some embodiments, aTWIST signaling inhibitor is a composition (e.g., an inhibitor bound toa delivery vehicle) described herein. In some embodiments, a TWISTsignaling inhibitor is a pharmaceutical composition described herein.

A “pharmaceutical composition” is a formulation containing thecomposition (e.g., an siRNA inhibitor, a small molecule inhibitor, or ansiRNA inhibitor bound to a nanoparticle) described herein in a formsuitable for administration to a subject. In embodiments, thepharmaceutical composition is in bulk or in unit dosage form. The unitdosage form is any of a variety of forms, including, for example, acapsule, an IV bag, a tablet, a single pump on an aerosol inhaler or avial. The quantity of active ingredient (e.g., an siRNA inhibitor, asmall molecule inhibitor, or an siRNA inhibitor bound to a nanoparticle)in a unit dose of composition is an effective amount and is variedaccording to the particular treatment involved. One skilled in the artwill appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In embodiments, the active TWIST signalinginhibitor is mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantsthat are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, anions, cations, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient. A thorough discussion of pharmaceutically acceptableexcipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (MackPub. Co., N.J. 1991). Pharmaceutically acceptable excipients intherapeutic compositions may contain liquids such as water, saline,glycerol and ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the compound of choice with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by intravenous infusion, orally,topically, intraperitoneally, intravesically or intrathecally.Parenteral administration, oral administration, and intravenousadministration are the preferred methods of administration. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

A pharmaceutical composition of the invention can be administered to asubject in many of the well-known methods currently used forchemotherapeutic treatment. For example, for treatment of cancers, acomposition of the invention may be injected directly into tumors,injected into the blood stream or body cavities or taken orally orapplied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not so high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an active composition (e.g., an anti-TWIST siRNA or anycomposition described herein). For example, described herein can be acancer monotherapy with one of compositions of the present invention toa subject in need of treatment of cancer. Monotherapy may be contrastedwith combination therapy, in which a combination of multiple activecompositions (e.g., multiple anti-TWIST siRNAs) is administered,preferably with each component of the combination present in atherapeutically effective amount. Monotherapy with a composition of thepresent invention may be more effective than combination therapy ininducing a desired biological effect.

As used herein, “combination therapy” or “co-therapy” includes theadministration of a composition of the present invention and at least asecond agent as part of a specific treatment regimen intended to providethe beneficial effect from the co-action of these therapeutic agents.The beneficial effect of the combination may include, but is not limitedto, pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected). “Combination therapy” may be, but generally isnot, intended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentinvention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, wherein each therapeuticagent is administered at a different time, as well as administration ofthese therapeutic agents, or at least two of the therapeutic agents, ina substantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

A composition of the present invention may be administered incombination with a second chemotherapeutic agent. The secondchemotherapeutic agent (also referred to as an anti-neoplastic agent oranti-proliferative agent) can be an alkylating agent; an antibiotic; ananti-metabolite; a detoxifying agent; an interferon; a polyclonal ormonoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histonedeacetylase inhibitor; a hormone; a mitotic inhibitor; an MTORinhibitor; a multi-kinase inhibitor; a serine/threonine kinaseinhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; ataxane or taxane derivative, an aromatase inhibitor, an anthracycline, amicrotubule targeting drug, a topoisomerase poison drug, an inhibitor ofa molecular target or enzyme (e.g., a kinase or a proteinmethyltransferase), a cytidine analogue drug or any chemotherapeutic,anti-neoplastic or anti-proliferative agent listed inwww.cancer.org/docroot/cdg/cdg_0.asp.

“Anti-cancer agent” is used in accordance with its plain ordinarymeaning and refers to a composition (e.g. compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In embodiments, ananti-cancer agent is a chemotherapeutic. In embodiments, an anti-canceragent is an agent identified herein having utility in methods oftreating cancer. In embodiments, an anti-cancer agent is an agentapproved by the FDA or similar regulatory agency of a country other thanthe USA, for treating cancer.

The anti-cancer agents set forth below are for illustrative purposes andnot intended to be limiting. The present invention includes at least oneanti-cancer agent selected from the lists below. The present inventioncan include more than one anti-cancer agent, e.g., two, three, four, orfive anti-cancer agents such that the composition of the presentinvention can perform its intended function.

In embodiments, the anticancer agent is a compound that affects histonemodifications, such as an HDAC inhibitor. In certain embodiments, ananticancer agent is selected from the group consisting ofchemotherapeutics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG,Abraxane™, Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-transretinoic acid, amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®,Camptosar®, CeeNU®, Clofarabine, Clolar™, Cytoxan®, daunorubicinhydrochloride, DaunoXome®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®,Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®,hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone,Ixempra®, L-asparaginase, Leukeran®, liposomal Ara-C, L-PAM, Lysodren,Matulane®, mithracin, Mitomycin-C, Myleran®, Navelbine®, Neutrexin®,nilotinib, Nipent®, Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®,Paraplatin®, Platinol®, prolifeprospan 20 with carmustine implant,Sandostatin®, Targretin@, Tasigna®, Taxotere®, Temodar®, TESPA,Trisenox®, Valstar®, Velban®, Vidaza™, vincristine sulfate, VM 26,Xeloda® and Zanosar®); biologics (such as Alpha Interferon, BacillusCalmette-Guerin, Bexxar®, Campath®, Ergamisol®, Erlotinib, Herceptin®,Interleukin-2, Iressa®, lenalidomide, Mylotarg®, Ontak®, Pegasys®,Revlimid®, Rituxan®, Tarceva™, Thalomid®, Tykerb®, Velcade® andZevalin™); corticosteroids, (such as dexamethasone sodium phosphate,DeltaSone® and Delta-Cortef®); hormonal therapies (such as Arimidex®,Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®,Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, Plenaxis™ andZoladex®); and radiopharmaceuticals (such as Iodotope®, Metastron®,Phosphocol® and Samarium SM-153).

In embodiments, the anti-cancer agent is a chemotherapeutic agent (alsoreferred to as an anti-neoplastic agent or anti-proliferative agent),selected from the group including an alkylating agent; an antibiotic; ananti-metabolite; a detoxifying agent; an interferon; a polyclonal ormonoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histonedeacetylase inhibitor; a hormone; a mitotic inhibitor; an MTORinhibitor; a multi-kinase inhibitor; a serine/threonine kinaseinhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; ataxane or taxane derivative, an aromatase inhibitor, an anthracycline, amicrotubule targeting drug, a topoisomerase poison drug, an inhibitor ofa molecular target or enzyme (e.g., a kinase or a proteinmethyltransferase), a cytidine analogue drug or any chemotherapeutic,anti-neoplastic or anti-proliferative agent listed inwww.cancer.org/docroot/cdg/cdg_0.asp.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary antibiotics include, but are not limited to, doxorubicin(Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone);bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal(DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin(Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin(Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodinel31 tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Exemplary Histone Deacetylase Inhibitors include, but are not limitedto, vorinostat (Zolinza).

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron);abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus Torisel); rapamune, ridaforolimus; orAP23573.

Exemplary multi-kinase inhibitors include, but are not limited to,sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474;PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol;Pkc412; bryostatin; KAI-9803; SF1126; or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);WHI-P154; WHI-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristine, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 Mpotassium oxonate), or lovastatin.

In embodiments, the anti-cancer agent is a chemotherapeutic agent or acytokine such as G-CSF (granulocyte colony stimulating factor).

In embodiments, the anti-cancer agents can be standard chemotherapycombinations such as, but not restricted to, CMF (cyclophosphamide,methotrexate and 5-fluorouracil), CAF (cyclophosphamide, adriamycin and5-fluorouracil), AC (adriamycin and cyclophosphamide), FEC(5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC(adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda(capecitabine), Cisplatin (CDDP), Carboplatin, TS-1 (tegafur, gimestatand otastat potassium at a molar ratio of 1:0.4:1), Camptothecin-11(CPT-11, Irinotecan or Camptosar™), CHOP (cyclophosphamide,hydroxydaunorubicin, oncovin, and prednisone or prednisolone), R-CHOP(rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisoneor prednisolone), or CMFP (cyclophosphamide, methotrexate,5-fluorouracil and prednisone).

In embodiments, the anti-cancer agents can be an inhibitor of an enzyme,such as a receptor or non-receptor kinase. Receptor and non-receptorkinases are, for example, tyrosine kinases or serine/threonine kinases.Kinase inhibitors described herein are small molecules, polynucleicacids, polypeptides, or antibodies. Exemplary kinase inhibitors include,but are not limited to, Bevacizumab (targets VEGF), BIBW 2992 (targetsEGFR and Erb2), Cetuximab/Erbitux (targets Erb1), Imatinib/Gleevic(targets Bcr-Abl), Trastuzumab (targets Erb2), Gefitinib/Iressa (targetsEGFR), Ranibizumab (targets VEGF), Pegaptanib (targets VEGF),Erlotinib/Tarceva (targets Erb1), Nilotinib (targets Bcr-Abl), Lapatinib(targets Erb1 and Erb2/Her2), GW-572016/lapatinib ditosylate (targetsHER2/Erb2), Panitumumab/Vectibix (targets EGFR), Vandetinib (targetsRET/VEGFR), E7080 (multiple targets including RET and VEGFR), Herceptin(targets HER2/Erb2), PKI-166 (targets EGFR), Canertinib/CI-1033 (targetsEGFR), Sunitinib/SU-11464/Sutent (targets EGFR and FLT3),Matuzumab/Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474(targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3),Vatalanib/Ptk787/ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614(targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322(targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606(targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154(targets JAK), WHI-P131 (targets JAK), sorafenib/Nexavar (targets RAFkinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-ß, KIT, FLT-3, and RET),Dasatinib/Sprycel (BCR/ABL and Src), AC-220 (targets Fit3), AC-480(targets all HER proteins, “panHER”), Motesanib diphosphate (targetsVEGF1-3, PDGFR, and c-kit), Denosumab (targets RANKL, inhibits SRC),AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, Rapamune (targets mTOR/FRAP1), Deforolimus (targets mTOR),Certican/Everolimus (targets mTOR/FRAP1), AP23573 (targets mTOR/FRAP1),Eril/Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK),Seliciclib/CYC202/Roscovitrine (targets CDK), SNS-032/BMS-387032(targets CDK), Ruboxistaurin (targets PKC), Pkc412 (targets PKC),Bryostatin (targets PKC), KAI-9803 (targets PKC), SF1126 (targets PI3K),VX-680 (targets Aurora kinase), Azdl 152 (targets Aurora kinase),Arry-142886/AZD-6244 (targets MAP/MEK), SCIO-469 (targets MAP/MEK),GW681323 (targets MAP/MEK), CC-401 (targets JNK), CEP-1347 (targetsJNK), and PD 332991 (targets CDK).

Additionally, the siRNA compound described herein can be co-administeredwith conventional immunotherapeutic agents including, but not limitedto, immunostimulants (e.g., Bacillus Calmette-Guérin (BCG), levamisole,interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g.,anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonalantibodies), immunotoxins (e.g., anti-CD33 monoclonalantibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.).

In a further embodiment, the siRNA compounds described herein can beco-administered with conventional radiotherapeutic agents including, butnot limited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y,⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(17m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

In embodiments, the anti-cancer agent used herein refers to doxorubicin,cisplatin, carboplatin, a taxane, camptothecin or any combinationthereof.

As used herein, a “subject in need thereof” or “a patient” is a subjecthaving cancer or a subject having a precancerous condition. Inembodiments, a subject in need thereof has cancer. In embodiments, asubject in need thereof has ovarian cancer. In embodiments, a subject inneed thereof has breast cancer. A “subject” or a “patient” includes amammal. The mammal can be e.g., a human or appropriate non-human mammal,such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep ora pig. The subject can also be a bird or fowl. In embodiments, themammal is a human. Thus the methods are applicable to both human therapyand veterinary applications.

In embodiments, a “subject in need thereof” is a subject that has breastcancer. Subjects with breast cancer includes subject with one or moresigns or symptoms of breast cancer. Signs and symptoms of breast cancerinclude lumps found in breast tissue, lumps found in the lymph nodes,thickening of breast tissue, one breast becoming larger or lower, anipple changing position or shape or becoming inverted, breast skinpuckering or dimpling, a rash on or around a nipple, discharge from oneor both nipples, constant pain in part of the breast or armpit, andswelling beneath the armpit or around the collarbone.

In embodiments, a “subject in need thereof” can also refer to a subjecthaving an increased risk of developing breast cancer relative to thepopulation at large. A subject with an increased risk of developingbreast cancer relative to the population at large is a female subjectwith a family history or personal history of breast cancer. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female subject having a germ-line orspontaneous mutation in BRCA1 or BRCA2, or both. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a female subject with a family history of breast cancer and agerm-line or spontaneous mutation in BRCA1 or BRCA2, or both. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female who is greater than 30 years old,greater than 40 years old, greater than 50 years old, greater than 60years old, greater than 70 years old, greater than 80 years old, orgreater than 90 years old. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is asubject with atypical hyperplasia of the breast, ductal carcinoma insitu (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS),lobular neoplasia, or a stage 0 growth or lesion of the breast (e.g.,stage 0 or grade 0 breast cancer, or carcinoma in situ).

In embodiments, a “subject in need thereof” is a subject that hasovarian cancer. Subjects with ovarian cancer includes subject with oneor more signs or symptoms of ovarian cancer. Signs and symptoms ofovarian cancer include bloating, pelvic or abdominal pain, troubleeating or feeling full quickly, urinary symptoms such as urgency orfrequency, fatigue, upset stomach, back pain, constipation, menstrualchanges, and/or abdominal swelling with weight loss.

In embodiments, a “subject in need thereof” can also refer to a subjecthaving an increased risk of developing ovarian cancer relative to thepopulation at large. A subject with an increased risk of developingovarian cancer relative to the population at large is a female subjectwith older age (e.g., 40 years old or older), obesity, no reproductivehistory, using fertility drug for longer than one year, using androgens,using estrogen therapy and hormone therapy, a family history or personalhistory of ovarian cancer, breast cancer or colorectal cancer.

In embodiments, a “subject in need thereof” has already undergone, isundergoing or will undergo, at least one therapeutic intervention forthe cancer or precancerous condition.

A subject in need thereof may have refractory cancer on most recenttherapy. “Refractory cancer” means cancer that does not respond totreatment. The cancer may be resistant at the beginning of treatment orit may become resistant during treatment. Refractory cancer is alsocalled resistant cancer. In some embodiments, the subject in needthereof has cancer recurrence following remission on most recenttherapy. In some embodiments, the subject in need thereof received andfailed all known effective therapies for cancer treatment. In someembodiments, the subject in need thereof received at least one priortherapy.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemias,lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas.Exemplary cancers that may be treated with a composition, pharmaceuticalcomposition, or method provided herein include lymphoma, sarcoma,bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer,esophageal cancer, gastric cancer, head and neck cancer, kidney cancer,myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g.triple negative, ER positive, ER negative, chemotherapy resistant,herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifenresistant, ductal carcinoma, lobular carcinoma, primary, metastatic),ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellularcarcinoma), lung cancer (e.g. non-small cell lung carcinoma, squamouscell lung carcinoma, adenocarcinoma, large cell lung carcinoma, smallcell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme,glioma, melanoma, prostate cancer, castration-resistant prostate cancer,breast cancer, triple negative breast cancer, glioblastoma, ovariancancer, lung cancer, squamous cell carcinoma (e.g., head, neck, oresophagus), colorectal cancer, leukemia, acute myeloid leukemia,lymphoma, B cell lymphoma, or multiple myeloma. Additional examplesinclude, cancer of the thyroid, endocrine system, brain, breast, cervix,colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung,melanoma, mesothelioma, ovary, sarcoma, stomach, uterus orMedulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiplemyeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,primary brain tumors, cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, endometrial cancer,adrenal cortical cancer, neoplasms of the endocrine or exocrinepancreas, medullary thyroid cancer, medullary thyroid carcinoma,melanoma, colorectal cancer, papillary thyroid cancer, hepatocellularcarcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, LobularCarcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells,cancer of the hepatic stellate cells, or prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with acomposition, pharmaceutical composition, or method provided hereininclude, for example, acute nonlymphocytic leukemia, chronic lymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a composition,pharmaceutical composition, or method provided herein include achondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma,osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma,fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma,Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma,immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma ofT-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parostealsarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma,synovial sarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a composition, pharmaceutical composition, or methodprovided herein include, for example, acral-lentiginous melanoma,amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo malignamelanoma, malignant melanoma, nodular melanoma, subungal melanoma, orsuperficial spreading melanoma.

“Breast cancer” includes all forms of cancer of the breast. Breastcancer can include primary epithelial breast cancers. Breast cancer caninclude cancers in which the breast is involved by other tumors such aslymphoma, sarcoma or melanoma. Breast cancer can include carcinoma ofthe breast, ductal carcinoma of the breast, lobular carcinoma of thebreast, undifferentiated carcinoma of the breast, cystosarcoma phyllodesof the breast, angiosarcoma of the breast, and primary lymphoma of thebreast. Breast cancer can include Stage I, II, IIIA, IIIB, IIIC and IVbreast cancer. Ductal carcinoma of the breast can include invasivecarcinoma, invasive carcinoma in situ with predominant intraductalcomponent, inflammatory breast cancer, and a ductal carcinoma of thebreast with a histologic type selected from the group consisting ofcomedo, mucinous (colloid), medullary, medullary with lymphcyticinfiltrate, papillary, scirrhous, and tubular. Lobular carcinoma of thebreast can include invasive lobular carcinoma with predominant in situcomponent, invasive lobular carcinoma, and infiltrating lobularcarcinoma. Breast cancer can include Paget's disease, Paget's diseasewith intraductal carcinoma, and Paget's disease with invasive ductalcarcinoma. Breast cancer can include breast neoplasms having histologicand ultrastructual heterogeneity (e.g., mixed cell types).

A breast cancer that is to be treated can include familial breastcancer. A breast cancer that is to be treated can include sporadicbreast cancer. A breast cancer that is to be treated can arise in a malesubject. A breast cancer that is to be treated can arise in a femalesubject. A breast cancer that is to be treated can arise in apremenopausal female subject or a postmenopausal female subject. Abreast cancer that is to be treated can arise in a subject equal to orolder than 30 years old, or a subject younger than 30 years old. Abreast cancer that is to be treated has arisen in a subject equal to orolder than 50 years old, or a subject younger than 50 years old. Abreast cancer that is to be treated can arise in a subject equal to orolder than 70 years old, or a subject younger than 70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA 15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich, or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both. A breast cancer that is to be treated canbe “triple-negative breast cancer” (TNBC) (estrogen receptor[ER]-negative, progesterone receptor [PR]-negative, and human epidermalgrowth factor receptor 2 [HER2]-negative).

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acomposition, pharmaceutical composition, or method provided hereininclude, for example, medullary thyroid carcinoma, familial medullarythyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocysticcarcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinomaof adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basalcell carcinoma, carcinoma basocellulare, basaloid carcinoma,basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolarcarcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, ductal carcinoma, carcinoma durum, embryonalcarcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinomaepitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere,carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giantcell carcinoma, carcinoma gigantocellulare, glandular carcinoma,granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ oranother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofcells in the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

“An effective amount” or “a therapeutically effective amount” asprovided herein refers to an amount effective to achieve its intendedpurpose. The actual amount effective for a particular application willdepend, inter alia, on the condition being treated. When administered inmethods to treat a disease, the pharmaceutical compositions describedherein will contain an amount of anti-TWIST siRNA and optionally atleast one anti-cancer agent to achieve the desired result, e.g.,reducing, eliminating, or slowing the progression of disease symptoms(e.g., cancer), or to exhibit a detectable therapeutic or inhibitoryeffect. The effect can be detected by any assay method known in the art.The precise effective amount for a subject will depend upon thesubject's body weight, size, and health; the nature and extent of thecondition; and the therapeutic or combination of therapeutics selectedfor administration. Therapeutically effective amounts for a givensituation can be determined by routine experimentation that is withinthe skill and judgment of the clinician. In a preferred aspect, thedisease or condition to be treated is cancer.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a composition of the presentinvention to alleviate the symptoms or complications of a disease,condition or disorder, or to eliminate the disease, condition ordisorder. The term “treat” can also include treatment of a cell in vitroor an animal model.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. The administration of compositions or pharmaceuticalcompositions of the invention may or can lead to the elimination of asign or symptom, however, elimination is not required. Effective dosagesshould be expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

Severity can also describe the degree to which a tumor has secretedgrowth factors, degraded the extracellular matrix, become vascularized,lost adhesion to juxtaposed tissues, or metastasized. Moreover, severitycan describe the number of locations to which a primary tumor hasmetastasized. Finally, severity can include the difficulty of treatingtumors of varying types and locations. For example, inoperable tumors,those cancers which have greater access to multiple body systems(hematological and immunological tumors), and those which are the mostresistant to traditional treatments are considered most severe. In thesesituations, prolonging the life expectancy of the subject and/orreducing pain, decreasing the proportion of cancerous cells orrestricting cells to one system, and improving cancer stage/tumorgrade/histological grade/nuclear grade are considered alleviating a signor symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body. For example, a cancer may also cause symptoms such asfever, fatigue, or weight loss. Pain may be an early symptom with somecancers such as bone cancers or testicular cancer. But most often painis a symptom of advanced disease. Along with cancers of the skin, someinternal cancers can cause skin signs that can be seen. These changesinclude the skin looking darker (hyperpigmentation), yellow (jaundice),or red (erythema); itching; or excessive hair growth.

Alternatively, or in addition, cancer subtypes present specific signs orsymptoms. Changes in bowel habits or bladder function could indicatecancer. Long-term constipation, diarrhea, or a change in the size of thestool may be a sign of colon cancer. Pain with urination, blood in theurine, or a change in bladder function (such as more frequent or lessfrequent urination) could be related to bladder or prostate cancer.

Changes in skin condition or appearance of a new skin condition couldindicate cancer. Skin cancers may bleed and look like sores that do notheal. A long-lasting sore in the mouth could be an oral cancer,especially in patients who smoke, chew tobacco, or frequently drinkalcohol. Sores on the penis or vagina may either be signs of infectionor an early cancer.

Unusual bleeding or discharge could indicate cancer. Unusual bleedingcan happen in either early or advanced cancer. Blood in the sputum(phlegm) may be a sign of lung cancer. Blood in the stool (or a dark orblack stool) could be a sign of colon or rectal cancer. Cancer of thecervix or the endometrium (lining of the uterus) can cause vaginalbleeding. Blood in the urine may be a sign of bladder or kidney cancer.A bloody discharge from the nipple may be a sign of breast cancer.

A thickening or lump in the breast or in other parts of the body couldindicate the presence of a cancer. Many cancers can be felt through theskin, mostly in the breast, testicle, lymph nodes (glands), and the softtissues of the body. A lump or thickening may be an early or late signof cancer. Any lump or thickening could be indicative of cancer,especially if the formation is new or has grown in size.

Indigestion or trouble swallowing could indicate cancer. While thesesymptoms commonly have other causes, indigestion or swallowing problemsmay be a sign of cancer of the esophagus, stomach, or pharynx (throat).

Recent changes in a wart or mole could be indicative of cancer. Anywart, mole, or freckle that changes in color, size, or shape, or losesits definite borders indicates the potential development of cancer. Forexample, the skin lesion may be a melanoma.

A persistent cough or hoarseness could be indicative of cancer. A coughthat does not go away may be a sign of lung cancer. Hoarseness can be asign of cancer of the larynx (voice box) or thyroid.

While the signs and symptoms listed above are the more common ones seenwith cancer, there are many others that are less common and are notlisted here.

Treating cancer may result in or can result in a reduction in size of atumor. A reduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size would be reduced byabout 5% or greater relative to its size prior to treatment; morepreferably, tumor size is reduced by about 10% or greater; morepreferably, reduced by about 20% or greater; more preferably, reduced byabout 30% or greater; more preferably, reduced by about 40% or greater;even more preferably, reduced by about 50% or greater; and mostpreferably, reduced by greater than about 75% or greater. Size of atumor may be measured by any reproducible means of measurement. The sizeof a tumor may be measured as a diameter of the tumor.

Treating cancer may result in or can result in a reduction in tumorvolume. Preferably, after treatment, tumor volume would be reduced byabout 5% or greater relative to its size prior to treatment; morepreferably, tumor volume is reduced by about 10% or greater; morepreferably, reduced by about 20% or greater; more preferably, reduced byabout 30% or greater; more preferably, reduced by about 40% or greater;even more preferably, reduced by about 50% or greater; and mostpreferably, reduced by greater than about 75% or greater. Tumor volumemay be measured by any reproducible means of measurement.

Treating cancer may result in or can result in a decrease in number oftumors. Preferably, after treatment, tumor number would be reduced byabout 5% or greater relative to number prior to treatment; morepreferably, tumor number is reduced by about 10% or greater; morepreferably, reduced by about 20% or greater; more preferably, reduced byabout 30% or greater; more preferably, reduced by about 40% or greater;even more preferably, reduced by about 50% or greater; and mostpreferably, reduced by greater than about 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer may result in or can result in a decrease in number ofmetastatic lesions in other tissues or organs distant from the primarytumor site. Preferably, after treatment, the number of metastaticlesions would be reduced by about 5% or greater relative to number priorto treatment; more preferably, the number of metastatic lesions isreduced by about 10% or greater; more preferably, reduced by about 20%or greater; more preferably, reduced by about 30% or greater; morepreferably, reduced by about 40% or greater; even more preferably,reduced by about 50% or greater; and most preferably, reduced by greaterthan about 75%. The number of metastatic lesions may be measured by anyreproducible means of measurement. The number of metastatic lesions maybe measured by counting metastatic lesions visible to the naked eye orat a specified magnification. Preferably, the specified magnification is2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer may result in or can result in an increase in averagesurvival time of a population of treated subjects in comparison to apopulation receiving carrier alone. Preferably, the average survivaltime would be increased by more than 30 days; more preferably, by morethan 60 days; more preferably, by more than 90 days; and mostpreferably, by more than 120 days. An increase in average survival timeof a population may be measured by any reproducible means. An increasein average survival time of a population may be measured, for example,by calculating for a population the average length of survival followinginitiation of treatment with an active composition. An increase inaverage survival time of a population may also be measured, for example,by calculating for a population the average length of survival followingcompletion of a first round of treatment with an active composition.

Treating cancer may result in or can result in an increase in averagesurvival time of a population of treated subjects in comparison to apopulation of untreated subjects. Preferably, the average survival timewould be increased by more than 30 days; more preferably, by more than60 days; more preferably, by more than 90 days; and most preferably, bymore than 120 days. An increase in average survival time of a populationmay be measured by any reproducible means. An increase in averagesurvival time of a population may be measured, for example, bycalculating for a population the average length of survival followinginitiation of treatment with an active composition. An increase inaverage survival time of a population may also be measured, for example,by calculating for a population the average length of survival followingcompletion of a first round of treatment with an active composition.

Treating cancer may result in or can result in increase in averagesurvival time of a population of treated subjects in comparison to apopulation receiving monotherapy with a drug that is not a compositionof the present invention. Preferably, the average survival time would beincreased by more than 30 days; more preferably, by more than 60 days;more preferably, by more than 90 days; and most preferably, by more than120 days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active composition. An increase in average survivaltime of a population may also be measured, for example, by calculatingfor a population the average length of survival following completion ofa first round of treatment with an active composition.

Treating cancer may result in or can result in a decrease in themortality rate of a population of treated subjects in comparison to apopulation receiving carrier alone. Treating cancer may result in or canresult in a decrease in the mortality rate of a population of treatedsubjects in comparison to an untreated population. Treating cancer mayresult in or can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a composition of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate would bedecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active composition. A decreasein the mortality rate of a population may also be measured, for example,by calculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active composition.

Treating cancer may result in or can result in a decrease in tumorgrowth rate. Preferably, after treatment, tumor growth rate would bereduced by at least 5% relative to number prior to treatment; morepreferably, tumor growth rate would be reduced by at least 10%; morepreferably, reduced by at least 20%; more preferably, reduced by atleast 30%; more preferably, reduced by at least 40%; more preferably,reduced by at least 50%; even more preferably, reduced by at least 50%;and most preferably, reduced by at least 75%. Tumor growth rate may bemeasured by any reproducible means of measurement. Tumor growth rate canbe measured according to a change in tumor diameter per unit time.

Treating cancer may result in or can result in a decrease in tumorregrowth. Preferably, after treatment, tumor regrowth would be less than5%; more preferably, tumor regrowth would be less than 10%; morepreferably, less than 20%; more preferably, less than 30%; morepreferably, less than 40%; more preferably, less than 50%; even morepreferably, less than 50%; and most preferably, less than 75%. Tumorregrowth may be measured by any reproducible means of measurement. Tumorregrowth is measured, for example, by measuring an increase in thediameter of a tumor after a prior tumor shrinkage that followedtreatment. A decrease in tumor regrowth is indicated by failure oftumors to reoccur after treatment has stopped.

II. Compositions

In one aspect, provided herein are TWIST signaling inhibitors. Inembodiments, TWIST signaling inhibitors described herein are TWISTinhibitors and/or inhibitors of TWIST signaling genes or protein. Inembodiments, TWIST signaling inhibitors provided herein include TWIST1inhibitor andTWIST1 signaling inhibitors. In embodiments, Inembodiments, TWIST signaling inhibitors provided herein include TWIST2inhibitors and TWIST2 signaling inhibitors. In embodiments, TWISTsignaling inhibitors provided herein include TWIST1 and TWIST2inhibitors and TWIST1 and TWIST2 signaling inhibitors. In embodiments,TWIST signaling inhibitors provided herein include TWIST inhibitors andinhibitors of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52) genes/proteins listed in Table 1 and one or more (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) genes/proteinsof Akt/PI3K signaling. In embodiments, TWIST signaling inhibitorsprovided herein include inhibitors of TWIST1, TWIST2, GAS6, L1CAM, PI3K,Akt, or any combination thereof.

In embodiments, TWIST signaling inhibitors provided herein are siRNAinhibitors. In embodiments, TWIST signaling inhibitors provided hereinare siRNAs against TWIST1, TWIST2, one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52) genes listed in Table 1 and Aktsignaling genes. In embodiments, TWIST signaling inhibitors providedherein are siRNAs against TWIST1, TWIST2, GAS6, L1CAM, PI3K, and/or Akt.

In embodiments, TWIST inhibitors are siRNA molecules (e.g., anti-TWISTsiRNAs). The siRNA molecule of the invention is an isolated siRNAmolecule that binds to a single stranded RNA molecule, which is amessenger RNA (mRNA) that encodes at least part of a peptide or proteinof TWIST1 or TWIST2.

TWIST1 and TWSIT2 are basic helix-loop-helix (bHLH) transcriptionfactors. TWIST1 and TWIST2 sequences are publicly available. Forexample, nucleotide sequence of TWSIST1 can be found at NM_000474.3: andnucleotide sequence of TWSIST1 can be found at: NP_000465.1. Forexample, amino acid sequences of TWIST2 can be found at NP_001258822.1:and amino acid sequences of TWIST2 can be found at NM_001271893.3.

One exemplary nucleic acid sequence of TWIST1 is provided below:

(SEQ ID NO: 13)GAGGTATAAGAGCCTCCAAGTCTGCAGCTCTCGCCCAACTCCCAGACACCTCGCGGGCTCTGCAGCACCGGCACCGTTTCCAGGAGGCCTGGCGGGGTGTGCGTCCAGCCGTTGGGCGCTTTCTTTTTGGACCTCGGGGCCATCCACACCGTCCCCTCCCCCTCCCGCCTCCCTCCCCGCCTCCCCCGCGCGCCCTCCCCGCGGAGGTCCCTCCCGTCCGTCCTCCTGCTCTCTCCTCCGCGGGCCGCATCGCCCGGGCCGGCGCCGCGCGCGGGGGAAGCTGGCGGGCTGAGGCGCCCCGCTCTTCTCCTCTGCCCCGGGCCCGCGAGGCCACGCGTCGCCGCTCGAGA

GCAGTCTCCAGGGGGATGCGCCCTGGTGAGGGGTGTGTGTGCGCGTGAGTGTGCGTGACAGGAGGGGAGACAGAGACACCCAGGGTCACGGGTAAGGACCGTTTTGTCAGCGCCACCCTTTCTTTCGGCTTTCAATTTTTGTTCTCCTTAAAACAAATGTTTTAAAACAAATTCCACCTCCTCCTCCTTTCCACCCACCCACTTCCTCTTGCCCTTGGGCTGAAATCCTTCCAGGTTGTTCAGCTTAATTTCTCAGTGGTGGTGATAAGAACAGTGCTCACTAGTCTTAGAAAACAGCCGCAGAGACCTAAACAATAACCGACTCCCCCCCCCCCCTCTGGGTTTTTGCAGATGTCATTGTTTCCAGAGAAGGAGAAAATGGACAGTCTAGAGACTCTGGAGCTGGATAACTAAAAATAAAAATATATGCCAAAGATTTTCTTGGAAATTAGAAGAGCAAAATCCAAATTCAAAGAAACAGGGCGTGGGGCGCACTTTTAAAAGAGAAAGCGAGACAGGCCCGTGGACAGTGATTCCCAGACGGGCAGCGGCACCATCCTCACACCTCTGCATTCTGATAGAAGTCTGAACAGTTGTTTGTGTTTTTTTTTTTTTTTTTTTTGACGAAGAATGTTTTTATTTTTATTTTTTTCATGCATGCATTCTCAAGAGGTCGTGCCAATCAGCCACTGAAAGGAAAGGCATCACTATGGACTTTCTCTATTTTAAAATGGTAACAATCAGAGGAACTATAAGAACACCTTTAGAAATAAAAATACTGGGATCAAACTGGCCTGCAAAACCATAGTCAGTTAATTCTTTTTTTCATCCTTCCTCTGAGGGGAAAAACAAAAAAAAACTTAAAATACAAAAAACAACATTCTATTTATTTATTGAGGACCCATGGTAAAATGCAAATAGATCCGGTGTCTAAATGCATTCATATTTTTATGATTGTTTTGTAAATATCTTTGTATATTTTTCTGCAATAAATAAATATAAAAAATTTAGAGAAUnderlined: transcription starting codesDouble underlined: translation starting codesWave underlined: bHLH domainItalic and underlined: stop codon

One skilled in the art will appreciate that TWIST (including TWIST1 andTWIST2) nucleic acid and protein molecules can vary from those publiclyavailable, such as polymorphisms resulting in one or more substitutions,deletions, insertions, or combinations thereof, while still retainingTWIST biological activity. Accordingly, in various embodiments, theamino acid sequence of the TWIST may be about 95%, about 96%, about 97%,about 98%, about 99% identical to the TWIST1 or TWIST2 sequence publiclyavailable, or fragment thereof. A fragment can be between 3-10 aminoacids, 10-20 amino acids, 20-40 amino acids, 40-56 amino acids in lengthor even longer. Amino acid sequences having about 95%, about 96%, about97%, about 98%, about 99% identity to the fragments described herein arealso included within the scope of the present invention.

In embodiments, the nucleic acid sequence of the TWIST may be about 95%,about 96%, about 97%, about 98%, about 99% identical to the TWIST1 orTWIST2 sequence publicly available, or fragment thereof. A fragment canbe between 3-10 nucleotides, 10-20 nucleotides, 20-40 nucleotides, 40-56nucleotides in length or even longer. Nucleic acid sequences havingabout 95%, about 96%, about 97%, about 98%, about 99% identity to thefragments described herein are also included within the scope of thepresent invention.

In embodiments, a TWIST signaling inhibitor is an anti-TWIST siRNA. Asused herein, the term “anti-TWIST siRNA” includes all forms ofanti-TWIST siRNA, including variants, modifications and derivativesthereof. In embodiments, the siRNA molecule is an oligonucleotide with alength of about 19 to about 35 base pairs (e.g., about 19, 20, 21, 22,23, 24, 25, 26 27, 28, 29, 30, 31, 32 33, 34, 35 base pairs). In anotherembodiment, the molecule is an oligonucleotide with a length of about 19to about 27 base pairs. In embodiments, the molecule is anoligonucleotide with a length of about 21 to about 25 base pairs. Inembodiments, the molecule may have blunt ends at both ends, or stickyends at both ends, or a blunt end at one end and a sticky end at theother. In embodiments, an anti-TWIST siRNA targets TWIST1 or variantsand homologues. In embodiments, an anti-TWIST siRNA targets TWIST2 orvariants and homologues. In embodiments, an anti-TWIST siRNA targetsboth TWIST1 and TWIST2 or their variants and homologues.

Exemplary anti-TWIST siRNA sequences include, but are not limited to:

Name Sequence siTWIST 419 Passenger: 5′-GGACAAGCUGAGCAAGAUU-3′(SEQ ID No: 1) Guide: 5′-AAUCUUGCUCAGCUUGUCCUU-3′ (SEQ ID No: 2)siTWIST494 Passenger: 5′-GCGACGAGCUGGACUCCAA-3′ (SEQ ID No: 3) Guide:5′-UUGGAGUCCAGCUCGUCGCUU-3′ (SEQ ID No: 4) TW511 Passenger:5′-GAUGGCAAGCUGCAGCUAUUU-3′ (SEQ ID No: 5) Guide:5′-AUAGCUGCAGCUUGCCAUCUU-3′ (SEQ ID No: 6) TW433 Passenger:5′-GAUUCAGACCCUCAAGCUGUU-3′ (SEQ ID No: 7) Guide:5′-CAGCUUGAGGGUCUGAAUCUU-3′ (SEQ ID No: 8) TW424 Passenger:5′-GCUGAGCAAGAUUCAGACCUU-3′ (SEQ ID No: 9) Guide:5′-GGUCUGAAUCUUGCUCAGCUU-3′ (SEQ ID No: 10)

One skilled in the art will appreciate that anti-TWIST siRNAs of theinvention also include sequences having about 95%, about 96%, about 97%,about 98%, about 99% identity to any one of SEQ ID No: 1-10.

As described above, antisense nucleic acids are capable of hybridizingto (e.g. selectively hybridizing to) a target nucleic acid (e.g. targetmRNA). In some embodiments, the antisense nucleic acid hybridizes to thetarget nucleic acid sequence (e.g. mRNA) under stringent hybridizationconditions. In some embodiments, the antisense nucleic acid hybridizesto the target nucleic acid (e.g. mRNA) under moderately stringenthybridization conditions.

An siRNA sequence (including antisense or sense sequence) may comprisenaturally occurring nucleotides or modified nucleotides. Examples ofsuch modifications include chemical substitutions at the ribose and/orphosphate and/or base positions. Modified nucleotides are described inU.S. Pat. No. 5,660,985, which describes oligonucleotides containingnucleotide derivatives chemically modified at the 2′ position of ribose,5 position of pyrimidines, and 8 position of purines. U.S. Pat. No.5,756,703 describes oligonucleotides containing various 2′-modifiedpyrimidines. U.S. Pat. No. 5,580,737 describes highly specific nucleicacid ligands containing one or more nucleotides modified with 2′-amino(2′-NH.sub.2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe)substituents.

Modifications of the siRNA sequences contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the siRNA bases or to the siRNA sequences as a whole.Such modifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, phosphorothioate or alkyl phosphate modifications,methylations, unusual base-pairing combinations such as the isobasesisocytidine and isoguanidine and the like. Modifications may alsoinclude 2′-O-methyl modifications, 2′-O-methyl modified ribose sugarswith terminal phosphorothioates and a cholesterol group at the 3′ end,2′-O-methoxyethyl (2′-MOE) modifications, 2′-fluoro modifications, and2′,4′ methylene modifications (referred to as “locked nucleic acids” orLNAs). Modifications can also include 3′ and 5′ modifications such ascapping.

In embodiments, antisense sequence (guide strand) and the sense(passenger strand) contain different modification(s). In embodiments,passenger strand may contain modifications that promote loading of theguide strand onto the mRNA cleavage machinery, modifications thatprevent the passenger sequence loading into RISC complex, modificationsthat prevent nuclease-mediated degradation, modifications that reduceimmunogenicity mediated by toll-like receptors and RIG-I, or anycombinations thereof. In embodiments, the passenger strand may containinverted abasic riboses, 2′-O-methyluracil, or combination thereof.Guide strand may contain modifications that increase their loadingefficiency to RISC complex. In embodiments, guide strand may contain2-thio-deoxyuracil. Exemplary chemically modified anti-TWIST siRNAsequences include, but are not limited to:

Name Sequence Modified Passenger:5′-iaBrGrGrArCrArArGrCrUrGrArGrCrArArGrAmUmUiaB-3′ siTWIST419(SEQ ID No: 11) iaB = inverted abasic ribose mU = 2′-O-methyluracilGuide: 5′-rArArUrCrUrUrGrCrUrCrArGrCrUrUrG2thio- dUrCrCrUrU-3′(SEQ ID No: 12) 2thio-dU = 2-thio-deoxyuracil

It is noted that the guide strand sequence of siTWIST419 has the fewestimmunogenic hits, after scanning for RNA sequence motifs that stimulateTLR8-dependent immune responses.

In embodiments, TWIST signaling inhibitors provided herein are one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) anti-GAS6 siRNAs. As used herein, the term“anti-GAS6 siRNA” includes all forms of anti-GAS6 siRNA, includingvariants, modifications and derivatives thereof. In embodiments, thesiRNA molecule is an oligonucleotide with a length of about 19 to about35 base pairs (e.g., about 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29,30, 31, 32 33, 34, 35 base pairs). In another embodiment, the moleculeis an oligonucleotide with a length of about 19 to about 27 base pairs.In still another embodiment, the molecule is an oligonucleotide with alength of about 21 to about 25 base pairs. In all of these embodiments,the molecule may have blunt ends at both ends, or sticky ends at bothends, or a blunt end at one end and a sticky end at the other.

In embodiments, TWIST signaling inhibitors provided herein are one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) anti-L1CAM siRNAs. As used herein, the term“anti-L1CAM siRNA” includes all forms of anti-L1CAM siRNA, includingvariants, modifications and derivatives thereof. In embodiments, thesiRNA molecule is an oligonucleotide with a length of about 19 to about35 base pairs (e.g., about 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29,30, 31, 32 33, 34, 35 base pairs). In another embodiment, the moleculeis an oligonucleotide with a length of about 19 to about 27 base pairs.In still another embodiment, the molecule is an oligonucleotide with alength of about 21 to about 25 base pairs. In all of these embodiments,the molecule may have blunt ends at both ends, or sticky ends at bothends, or a blunt end at one end and a sticky end at the other.

A siRNA sequence against a specific gene can be designed according toany method known in the art. In embodiments, siRNAs against GAS6, L1CAM,and HMGA2 were obtained from Santa Cruz Biotechnology (Dallas, Tex.;item numbers sc-35450, sc-43172, and sc-37994, respectively).

In embodiments, TWIST signaling inhibitors provided herein are smallmolecule inhibitors. In embodiments, TWIST signaling inhibitors providedherein are small molecule inhibitors against TWIST1, TWIST2, one or moreproteins listed in Table 1 and/or one or more proteins of Akt/PI3Ksignaling. In embodiments, TWIST signaling inhibitors provided hereininclude a plurality of small molecule inhibitors. In embodiments, TWISTsignaling inhibitors provided herein are small molecule inhibitorsagainst TWIST1, TWIST2, GAS6, L1CAM, PI3K, and/or Akt. In embodiments,small molecule inhibitors against PI3K/Akt signaling include, but arenot limited to, Wortmannin, demethoxyviridin, LY294002, perifosine,idelalisib, buparlisib (BKM 120), duvelisib, alpelisib (BYL719), TGR1202, copanlisib (BAY 80-6946), PX-866, dactolisib, RP6530, SF1126,INK1117, pictilisib, XL147 (also known as SAR245408), XL765 (also knownas SAR245409), palomid 529, GSK1059615, ZSTK474, PWT33597, CUDC-907,ME-401, IPI-549, IC87114, TG100-115, CAL263, RP6503, PI-103, GNE-477,and AEZS-136.

In embodiments, TWIST signaling inhibitors provided herein include oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more) siRNA inhibitors and one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more)small molecule inhibitors. In embodiments, TWIST signaling inhibitorsprovided herein include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) anti-TWIST siRNAsand/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more) anti-GAS6 siRNAs and/or one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more) anti-L1CAM siRNAs and/or one or more (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) PI3Ksmall molecule inhibitors and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) Akt smallmolecule inhibitors.

The invention also provides a composition that includes a TWISTsignaling inhibitor described herein bound to a delivery vehicle.

In embodiments, the delivery vehicle is a nanoparticle. In embodiments,the delivery vehicle is a lipid particle (or lipid vehicle). The term“delivery vehicle” or “carrier” refers to any support structure thatbrings about the transfer of a component of genetic material or aprotein. Genetic material includes but is not limited to DNA, RNA orfragments thereof and proteins or polypeptides comprise amino acids andinclude but are not limited to antigens, antibodies, ligands, receptorsor fragments thereof. Delivery vehicles include but are not limited tovectors such as viruses (examples include but are not limited toretroviruses, adenoviruses, adeno-associated viruses, pseudotypedviruses, replication competent viruses, herpes simplex virus), viruscapsids, liposomes or liposomal vesicles, lipoplexes, polyplexes,dendrimers, macrophages, artificial chromosomes, nanoparticles, polymersand also hybrid particles, examples of which include virosomes. Deliveryvehicles may have multiple surfaces and compartments for attachment andstorage of components. These include but are not limited to outersurfaces and inner compartments.

In embodiments, the delivery vehicle is a nanoparticle or a lipidparticle or a viral vector. Any nanoparticles known for siRNA/smallmolecule delivery can be used for the invention described herein. Recentdramatic advances in nanotechnology have led to the development of avariety of nanoparticles (NPs) that provide valuable tools. Numerousnanomaterials such as polymers, liposomes, protein based NPs andinorganic NPs have been developed and a variety of particles arecurrently being evaluated in clinical studies with promising initialresults; and some liposomal NPs are approved by the FDA. One of themajor advantages of using these NPs is that they offer targetedtissue/site delivery. Their small size allows NPs to escape throughblood vessels at the tissue site through the leaky vascular structure(Enhanced permeability and retention effect). In addition to thispassive mechanism, a variety of targeting moieties can be attached toNPs to confer active targeting capability. The ability of nanoparticlesto target delivery of anticancer drugs to tumors also results indecreased chemotherapy-related off-target toxicity for patients.Exemplary nanoparticles that can be used for delivering compositionsdescribed herein include, but are not limited to, solid nanoparticles(e.g., metals such as silver, gold, iron, titanium), non-metal,lipid-based solids (e.g., liposome), polymers (e.g., polyethylenimene,dendrimer), suspensions of nanoparticles, or combinations thereof (e.g.,polyethylenimene-liposome, dendrisome). Any compositions describedherein (such as Mito-Cas9, mito-Cpf1, or other mito-RNA guided nucleases(mito-RGN)) may be delivered in nanopoarticle complexes in the form ofprotein, DNA, or mRNA. Additional information about nanoparticles thatcan be used by the compositions described herein can be found in Coelhoet al., N Engl J Med 2013; 369:819-29, Tabernero et al., CancerDiscovery, April 2013, Vol. 3, No. 4, pages 363-470, Zhang et al.WO2015089419 A2, and Zuris J A et al., Nat Biotechnol. 2015;33(1):73-80, each of which is incorporated herein by reference.

In embodiments, the nanoparticle used herein is mesoporous silicananoparticle (MSN). MSNs are inorganic NPs that have developed as adelivery system for anticancer drugs and siRNA (silencing smallinterfering RNA). MSNs are synthesized by the sol-gel method, whichenables preparation of homogeneous particles with diameters as small as40 nm or as large as desired. They contain thousands of pores thatprovide a large storage space for drugs and other reagents. Thesenanomaterials are biocompatible and their safety has been demonstratedin a number of animal experiments. MSNs are taken up by endocytosis,localized first to lysosomes, deliver drugs, and are eventuallyexocytosed out of cells. MSNs can be coated with targeting moieties thatbind to receptors on cancer cells, thereby greatly enhancing particleuptake, and with valves that control the release of drug cargo resultingin apoptosis. Additional information about the making and use of MSNscan be found in the U.S. Patent Publication 2012/0207795, the content ofwhich is incorporated herein as entirety.

In embodiments, the surface of the nanoparticle (e.g. MSNs) arechemically modified. To maximize the delivery of negatively chargednucleic acids to cells, the silica surface may be converted intopositive charge in order to bind DNA and siRNA. Some of the methods forintroducing cationic charge on inorganic materials, which includesilica, iron oxide, and gold, typically involve surface grafting withamine groups and coating with cationic polymers (e.g. polyethyleneimine,polyamidoamine, polylysine) through either covalent or non-covalent(e.g. electrostatic) association (Radu et al., J. Am. Chem. Soc., vol.126, pp. 13216-13217, 2004; Bharali et al., Proc. Natl. Acad. Sci.U.S.A., vol., 102, pp. 11539-11544, 2005; Bonoiu et al., Proc. Natl.Acad. Sci. U.S.A., vol. 106, pp. 5546-5550, 2009; Elbakry et al., NanoLett., vol. 9, pp. 2059-2064, 2009; Fuller et al., Biomaterials, vol.29, pp. 1526-1532, 2008; Kneuer et al., Bioconjugate Chem., vol. 11, pp.926-932, 2000; McBain et al., J. Mater. Chem., vol. 17, pp. 2561-2565,2007; Zhu et al., Biotechnol. Appl. Biochem., vol. 39, pp. 179-187,2004). In embodiments, the nanoparticle (e.g. MSN) is bound topolyethyleneimine (PEI). The PEI may be referred to herein as anon-covalent linker.

As used herein, the term “bioconjugate” or “bioconjugate reactive group”or “bioconjugate linker” refers to the association between atoms ormolecules. The association can be direct or indirect. For example, aconjugate between a first moiety (e.g. —NH₂, —COOH,—N-hydroxysuccinimide, or -maleimide) and a second moiety (e.g.,sulfhydryl, sulfur-containing amino acid) provided herein can be direct,e.g., by covalent bond or linker (e.g. a first linker of second linker),or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions(e.g. ionic bond, hydrogen bond, halogen bond), van der Waalsinteractions (e.g. dipole-dipole, dipole-induced dipole, Londondispersion), ring stacking (pi effects), hydrophobic interactions andthe like). In embodiments, conjugates are formed using conjugatechemistry including, but are not limited to nucleophilic substitutions(e.g., reactions of amines and alcohols with acyl halides, activeesters), electrophilic substitutions (e.g., enamine reactions) andadditions to carbon-carbon and carbon-heteroatom multiple bonds (e.g.,Michael reaction, Diels-Alder addition). These and other usefulreactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982. In embodiments, thefirst moiety (e.g., a TWIST signaling inhibitor) is non-covalentlyattached to the second moiety on the nanoparticle through a non-covalentchemical linker or covalent chemical linker formed by a reaction betweena component of the first moiety (e.g., a TWIST signaling inhibitor) anda component of the second moiety on the delivery vehicle (e.g.,nanoparticle or lipid particle). In embodiments, the first moiety (e.g.,a TWIST signaling inhibitor) includes one or more reactive moieties,e.g., a covalent reactive moiety, as described herein (e.g., alkyne,azide, amine, ester, N-hydroxy-succinimide, maleimide or thiol reactivemoiety). In embodiments, the first moiety (e.g., a TWIST signalinginhibitor) includes a linker (e.g., first linker) with one or morereactive moieties, e.g., a covalent reactive moiety, as described herein(e.g., alkyne, azide, amine, ester, N-hydroxy-succinimide, maleimide orthiol reactive moiety). In embodiments, the delivery vehicle (e.g.,nanoparticle or lipid particle) includes one or more reactive moieties,e.g., a covalent reactive moiety, as described herein (e.g., alkyne,azide, amine, ester, N-hydroxy-succinimide, maleimide or thiol reactivemoiety). In embodiments, the delivery vehicle (e.g., nanoparticle orlipid particle) includes a linker with one or more reactive moieties,e.g., a covalent reactive moiety, as described herein (e.g., alkyne,azide, amine, ester, N-hydroxy-succinimide, maleimide or thiol reactivemoiety).

Useful reactive functional groups (e.g., reactive groups such asbioconjugate or bioconjugate reactive groups) used for conjugatechemistries herein include, for example:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups which can be converted to esters, ethers,        aldehydes, etc.    -   (c) haloalkyl groups wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the site of the halogen atom;    -   (d) dienophile groups which are capable of participating in        Diels-Alder reactions such as, for example, maleimido or        maleimide groups;    -   (e) aldehyde or ketone groups such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be converted to disulfides, reacted        with acyl halides, or bonded to metals such as gold, or react        with maleimides;    -   (h) amine or sulfhydryl groups (e.g., present in cysteine),        which can be, for example, acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds;    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis;    -   (l) metal silicon oxide bonding; and    -   (m) metal bonding to reactive phosphorus groups (e.g.        phosphines) to form, for example, phosphate diester bonds.    -   (n) azides coupled to alkynes using copper catalyzed        cycloaddition click chemistry.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theconjugate described herein. Alternatively, a reactive functional groupcan be protected from participating in the crosslinking reaction by thepresence of a protecting group. In embodiments, the bioconjugatecomprises a molecular entity derived from the reaction of an unsaturatedbond, such as a maleimide, and a sulfhydryl group.

In embodiments, the surface of the nanoparticle (e.g. MSNs) are bound(e.g., coated) with positively charged compound or chemical moiety (e.g.polyethyleneimine (PEI)), which non-covalently binds to negativelycharged nucleic acid (e.g., anti-TWIST signaling siRNA) viaelectrostatic interaction. In embodiments the positively chargedcompound (also referred to herein a a positively charged non-covalentlinker) is a synthetic cationic polymer. In embodiments, the positivelycharged non-covalent linker compacts DNA and siRNA into complexes thatare effectively taken up in cells. In embodiments, the non-covalentlinker (e.g. positively charged non-covalent linker) is attached tonanoparticle surfaces through covalent (e.g. using boiconjugatetechniques as disclosed herein and known in the art) and electrostaticinteractions. For example, PEIs are synthetic cationic polymers thatcompact DNA and siRNA into complexes that are effectively taken up incells. In embodiments, the PEI is attached to nanoparticle surfacesthrough covalent and electrostatic interactions. Several PEI polymersizes ranging from MW of 0.6 to 25 KD can be used in the invention. Thesurface of nanoparticle (e.g. MSNs) may be modified using low molecularweight multi-branched PEI (e.g., less than or equal to MW 1200) due totheir low toxicity.

In embodiments, the nanoparticle (e.g. MSNs) contain one or morenanodevices called “nanovalves.” Nanovalves are made of chemicalmoieties (e.g. rotaxanes and pseudorotaxanes) that include a stalk and amoving part. The moving part acts as a gatekeeper of the pores'openings. The opening can be triggered by recognition events (pH, redox,charge, metal ions and biomolecules such as enzymes) and externalcontrol (magnetic field, light). One type of nanovalve is triggered bylow pH (around pH 6). This consists of a stalk that has cyclodextrinattached. The cyclodextrin binding causes the nanovalve to be closed.Upon exposure to low pH (such as in tumor tissue), cyclodextrin comesoff thus releasing the contents of the pores. The pH threshold can beadjusted by chemically modifying the valve. Additional information aboutnanovalve that is triggered by low pH can be found in the U.S. PatentPublication 2010/0310465, the content of which is incorporated herein asentirety.

In embodiments, the nanoparticle (e.g. MSNs) have nanovalves that areactivated by an external stimulus. For example, the MSNs include thermosensitive nanovalves. Nanoparticles (such as MSNs) containing nanovalvesthat are activated by an external stimulus are an attractive method fordrug delivery as they provide non-invasive treatment that has refinedcontrol over a selected area, the exposure time, and hence the dosage.In some embodiments, magnetic core-shell nanoparticles (e.g. MSNs suchas mag@MSN or Magnet-MSN, referred to herein as MSN-B) are utilized. Forexample, when exposed to an oscillating magnetic field, the magneticnanocrystal (NC) cores produce heat (T>42° C.) that can be used tostimulate a thermally responsive nanovalve (note that the nanovalve ismade up of a thermo sensitive pseudorotaxane). Additional informationabout MSN-B can be found in the U.S. Patent Publication 2010/0255103,the content of which is incorporated herein as entirety. Additionalinformation about nanovalve that is triggered by light can be found inthe U.S. Patent Publication 2010/0284924, the content of which isincorporated herein as entirety.

In embodiments, the nanoparticle is porous. The porous structure ofnanoparticles (e.g. MSNs) may allow both the binding of nucleotides onthe surface as well as the encapsulation of small molecules within theparticles. In embodiments, one or more anti-cancer agents areencapsulated in within the pores of the nanoparticles (e.g. MSNs) thathave nanovalves. The release of the anti-cancer agent may be controlledby nanovalves within the nanoparticle (e.g. MSNs). In particular,nanovalves may provide an open/close function so that anti-cancer agentstored in the pores of the nanoparticles (e.g. MSNs) can only bereleased when they encounter conditions (such as low pH, magnetic field,light) that allow nanovalves to open.

In embodiments, the nanoparticle (e.g. MSN) has surface modificationsincluding the attachment of tumor targeting moieties. In embodiments,the nanoparticle (e.g. MSN) have surface modifications including theattachment of PEI and tumor targeting moieties. In some embodiments, thetumor targeting moiety is hyaluronic acid (HA).

In embodiments, composition of the invention includes an anti-TWISTsignaling siRNA described herein bound to MSN via PEI that is attachedto MSN surface (PEI-MSN). In embodiments, such PEI-MSN also includesnanovavles (low pH, magnetic field, or light triggered). In embodiments,such PEI-MSN containing nanovalves also encapsulates one or moreanti-cancer agents.

In embodiments, composition of the invention includes an anti-TWISTsignaling siRNA described herein bound to PEI-MSN that also has HAattached to its surface (PEI-MSN-HA). In embodiments, such PEI-MSN-HAalso includes nanovavles (low pH, magnetic field, or light triggered).In embodiments, such PEI-MSN-HA containing nanovalves also encapsulatesone or more anti-cancer agents.

In embodiments, an anti-TWIST signaling siRNA described herein is boundto a dendrimer-based nanoparticle. In embodiment, the nanoparticle isYTZ3-15 dendrimer. In embodiment, the nanoparticle is polyamidoaminedendrimer. Dendrimers are repetitively branched molecules. Nanoparticlesbased on the dendritic polymer or dendrimer are referred asdendrimer-based nanoparticles. The chemical formula for each YTZ3-15dendrimer is C₁₂₅H₂₄₇N₃₇O₂₀ with a molecular weight of 2586.9448Daltons. They are formed by click chemistry and consist of two lipidtails at one end and a dendron with eight terminal amines on theopposite end. The dendrimer was purified by column chromatography onsilica gel with Petroleum ether/EtOAc. These dendrimers spontaneouslyaggregate to form micelles ranging in size from 100 nm to 200 nm. Whenin the presence of siRNA, these dendrimers rearrange into smaller (6-8nm) substructures as part of the larger micelles in order to allow formore electrostatic interactions between the negatively charged siRNA andthe positively charged amines on the dendrimer (see FIG. 10).

In embodiments, a dendrimer-based nanoparticle bound (covalently ornon-covalently) to an anti-TWIST signaling siRNA described herein may befurther bound (covalently or non-covalently) to an anti-cancer agent.

In embodiments, an anti-cancer agent used herein is doxorubicin,cisplatin, carboplatin, a taxane, camptothecin or any combinationthereof.

In embodiments, the delivery vehicle used herein is a vector. Inembodiments, the vector is a replication-incompetent viral vector. Forexample, the replication-incompetent viral vector is areplication-incompetent DNA viral vector (including, but is not limitedto, adenoviruses, adeno-associated viruses). For example, thereplication-incompetent viral vector is a replication-incompetent RNAviral vector (including, but is not limited to, replication defectiveretroviruses, lentiviruses, and rabies viruses).

In embodiments, the delivery vehicle used herein is a lipid particle—aparticle having lipid as a component, such as liposomes or liposomalvesicles or lipoplexes. Liposomes, also known as vesicles, are generallycomposed of phospholipids and other lipid components such ascholesterol. They can function as carriers whose essential structuralfeature is a bipolar lipid membrane which envelops an aqueous corevolume in which pharmacological agents are solubilized and thereforeencapsulated. Various lipid formulations and methods for theirpreparation have been described for the delivery of pharmaceuticallyactive agents to a host. For example, Geho and Lau in U.S. Pat. No.4,603,044 describe a targeted liposomal delivery system for delivery ofa drug to the hepatobiliary receptors of the liver. The system iscomposed of a drug or diagnostic agent encapsulated in or associatedwith lipid membrane structures in the form of vesicles or liposomes, anda molecule having a fatty substituent attached to the vesicle wall and atarget substituent which is a biliary attracted chemical, such as asubstituted iminodiacetate complex. The system is particularly usefulfor the delivery of insulin and serotonin in the treatment of Types Iand II diabetes, respectively. Several cationic lipid reagents havebecome commercially available for transfecting eukaryotic cells. Theseexamples include Lipofectin® (DOTMA:DOPE) (Invitrogen, Carlsbad,Calif.), LipofectAmine™ (DOSPA:DOPE)(Invitrogen). LipofectAmine2000™(Invitrogen), LipofectAmine 3000™ (Invitrogen), Lipofectamine RNAiMax™(Invitrogen). Lipofectamme LTX™ (Thermo Fisher Scientific), Fugene®,Transfectam® (DOGS), Effectene®, DC-Chol. US Patent Publication No.20050019923 involves cationic dendrimers for delivering bioactivemolecules, such as polynucleotide molecules, peptides and polypeptidesand/or pharmaceutical agents, to a mammalian body, given the lowtoxicity and targeting specificity. Other derivatives of cationicdendrimer mentioned in Bioactive Polymers, US published application20080267903, may also be suitable delivery vehicles for mitoCas9 genetherapy.

Various polymeric formulations of biologically active agents and methodsfor their preparation have been described. U.S. Pat. Nos. 3,773,919,3,991,776, 4,076,779, 4,093,709, 4,118,470, 4,131,648, 4,138,344,4,293,539 and 4,675,189, inter alia, disclose the preparation and use ofbiocompatible, biodegradable polymers, such as poly (lactic acid),poly(glycolic acid), copolymers of glycolic and lactic acids, poly(o-hydroxycarboxy lie acid), polylactones, polyacetals, polyorthoestersand polyorthocarbonates, for the encapsulation of drugs and medicaments.These polymers mechanically entrap the active constituents and laterprovide controlled release of the active ingredient via polymerdissolution or degradation. Certain condensation polymers formed fromdivinyl ethers and polyols are described in Polymer Letters, 18, 293(1980). Polymers have proven to be successful controlled-release drugdelivery devices.

More information about liposomal constructs or polymeric constructs thatcan be used for the present invention can be found at Schwendener R A etal., Ther Adv Vaccines. 2014 November; 2(6): 159-182; Li Y et al., JGene 2011, Med 13: 60-72; Pichon C et al., Methods Mol Biol 2013 969:247-274; McNamara M A et al., J Immunol Res. 2015; 2015: 794528; SayourE. J. et al., Journal for Immunotherapy of Cancer. 2015; 3, article 13;Bettinger T. et al, Current Opinion in Molecular Therapeutics. 2001;3(2):116-124; Lu D. et al., Cancer Gene Therapy. 1994; 1(4):245-252;Wasungu L. et al., Journal of Controlled Release. 2006; 116(2):255-264;Little S. et al., Proceedings of the National Academy of Sciences of theUnited States of America. 2004; 101(26):9534-9539; Phua K. et al.,Journal of Controlled Release. 2013; 166(3):227-233; Su X et al.,Molecular Pharmaceutics. 2011; 8(3):774-787; Phua K. K. L. et al.,Nanoscale. 2014; 6(14):7715-7729; Phua K. K. L. et al., ScientificReports. 2014; 4, article 5128, the content of each of which isincorporated herein as entirety.

The present invention also provides a DNA sequence encoding ananti-TWIST siRNA sequence of any one of SEQ ID Nos: 1-10. Such DNAsequence can be further included to a vector for transfection andexpression of the anti-TWIST siRNA.

The present invention further provides pharmaceuticalcompositions/formulations that include a composition disclosed herein incombination with at least one pharmaceutically acceptable excipient orcarrier.

Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, or acetate at a pH typically of 5.0to 8.0, most often 6.0 to 7.0; salts such as sodium chloride, potassiumchloride, etc. to make isotonic; antioxidants, preservatives, lowmolecular weight polypeptides, proteins, hydrophilic polymers such aspolysorbate 80, amino acids such as glycine, carbohydrates, chelatingagents, sugars, and other standard ingredients known to those skilled inthe art (Remington's Pharmaceutical Science 16^(th) edition, Osol, A.Ed. 1980).

A pharmaceutical formulation including a composition as described hereincan be administered by a variety of methods known in the art. The routeand/or mode of administration vary depending upon the desired results.In embodiments, administration is intravenous, intramuscular,intraperitoneal, or subcutaneous, or administered proximal to the siteof the target. Pharmaceutically acceptable excipients can be suitablefor intravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

Pharmaceutical formulations of the nucleic acid as described herein canbe prepared in accordance with methods well known and routinelypracticed in the art. See, e.g., Remington: The Science and Practice ofPharmacy, Mack Publishing Co., 20^(th) ed., 2000; and Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978. Pharmaceutical compositions are preferablymanufactured under GMP conditions.

Actual dosage levels of the active ingredients (i.e., the compositionsdescribed herein) in the pharmaceutical compositions of the presentinvention can be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel depends upon a variety of pharmacokinetic factors including theactivity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular composition (e.g., the nucleic aciddescribed herein) being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician or veterinarian can start doses of the nucleic acid (e.g.,anti-TWIST siRNA) of the invention employed in the pharmaceuticalformulation at levels lower than that required to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. In general, effective doses of the compositions ofthe present invention vary depending upon many different factors,including the specific disease or condition to be treated, means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. For administration witha pharmaceutical formulation of the invention, the dosage ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once per every two weeks or once a monthor once every 3 to 6 months.

The compositions provided herein can be administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring immuneresponse to the neo-antigen. Alternatively, composition can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the composition in the patient. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

A composition or a pharmaceutical composition provided herein may, ifdesired, be presented in a kit (e.g., a pack or dispenser device) whichmay contain one or more unit dosage forms containing the composition orthe pharmaceutical composition, for example (1) a TWIST signalinginhibitor, (2) a composition including a TWIST signaling inhibitor boundto a delivery vehicle, (3) a composition including an anti-cancer drugand a TWIST signaling inhibitor that is optionally bound to a deliveryvehicle, or (4) a pharmaceutical composition including apharmaceutically acceptable excipient and a composition describedherein. The pack may for example comprise metal or plastic foil, such asa blister pack. The pack or dispenser device may be accompanied byinstructions for administration. Compositions comprising a compositiondescribed herein formulated in a compatible pharmaceutical carrier mayalso be prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition. Instructions for use may also beprovided.

Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying outthe assay may be included in the kit. The assay may for example be inthe form of q-PCR, Western Blot analysis, Immunohistochemistry (IHC),immunofluorescence (IF), sequencing and Mass spectrometry (MS) as knownin the art.

III. Methods

The invention further provides methods of using the compositionsdescribed herein.

In one aspect, the invention relates to a method of reversing to ananti-cancer drug (e.g., doxorubicin, cisplatin, carboplatin, a taxane,camptothecin or any combination thereof) in a subject by administering atherapeutically effective amount of a TWIST signaling inhibitordescribed herein to the subject.

In one aspect, the invention relates to a method of treating cancer in asubject in need thereof by administering to the subject atherapeutically effective amount of a TWIST signaling inhibitordescribed herein.

In another aspect of the invention relates to a method of inhibitingmetastasis in a subject in need thereof by administering to the subjecta therapeutically effective amount of a TWIST signaling inhibitordescribed herein.

In embodiments, a subject in need thereof of the method described hereinmay be resistant to an anti-cancer drug (e.g., doxorubicin, cisplatin,carboplatin, a taxane, camptothecin or any combination thereof).

In embodiments, any method described herein may also includeadministering to the subject an effective amount of an anti-canceragent, simultaneously as the TWSIT signaling inhibitor or subsequently(after the administration of the TWIST signaling inhibitor). Ananti-cancer agent can be any anti-cancer agent described herein. Inembodiments, an anti-cancer agent is doxorubicin, cisplatin,carboplatin, a taxane, camptothecin or any combination thereof.

In embodiments, the TWIST signaling inhibitor used in any methoddescribed herein is a TWIST inhibitor described herein. In embodiments,the TWIST signaling inhibitor is a TWIST1 signaling inhibitor. Inembodiments, the TWIST signaling inhibitor is a TWIST2 signalinginhibitor. In embodiments, the TWIST signaling inhibitor is a TWIST1 andTWIST2 signaling inhibitor. In embodiments, TWIST signaling inhibitorsinclude inhibitors of TWIST1, TWIST2, one or more genes/proteins listedin Table 1 and Akt signaling genes/proteins. In embodiments, TWISTsignaling inhibitors include inhibitors of TWIST1, TWIST2, GAS6, L1CAM,PI3K, Akt, or any combination thereof. In embodiments, the TWISTsignaling inhibitor used in any method described herein includes aplurality of TWIST signaling inhibitors provided herein.

In embodiments, TWIST signaling inhibitors used in any method describedherein are siRNA inhibitors. In embodiments, TWIST signaling inhibitorsare siRNAs against TWIST1, TWIST2, one or more genes listed in Table 1and Akt signaling genes. In embodiments, TWIST signaling inhibitors aresiRNAs against TWIST1, TWIST2, GAS6, L1CAM, PI3K, and/or Akt. A siRNAsequence against a specific gene can be designed according to any methodknown in the art. In embodiments, the siRNA inhibitor used in any methoddescribed herein includes a plurality of siRNAs (i.e., a pooled siRNAs)against TWIST1, TWIST2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52) of the genes listed in Table 1 and Aktsignaling genes.

In embodiments, the TWIST signaling inhibitor used in any methoddescribed herein is a TWIST1 inhibitor. In embodiments, the TWISTsignaling inhibitor used in any method described herein is an anti-TWISTsiRNA. In embodiments, the TWIST signaling inhibitor used in any methoddescribed herein is an anti-TWIST siRNA having a sequence of any one ofSEQ ID Nos: 1-12. In embodiments, the TWIST signaling inhibitor used inany method described herein is a composition including a nanoparticlebound with an anti-TWIST siRNA. In embodiments, the TWIST signalinginhibitor used in any method described herein is a pharmaceuticalcomposition described herein.

In embodiments, pooled siRNAs against GAS6, L1CAM, and HMGA2 wereobtained from Santa Cruz Biotechnology (Dallas, Tex.; item numberssc-35450, sc-43172, and sc-37994, respectively).

In embodiments, TWIST signaling inhibitors are small moleculeinhibitors. In embodiments, TWIST signaling inhibitors are smallmolecule inhibitors against one or more proteins listed in Table 1 andAkt/PI3K signaling proteins. In embodiments, TWIST signaling inhibitorsare small molecule inhibitors against TWIST1, TWIST2, GAS6, L1CAM, PI3K,and/or Akt. In embodiments, the small molecule inhibitor used in anymethod described herein includes a plurality of small moleculeinhibitors (i.e., a pooled small molecule inhibitors) against one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) ofTWIST1, TWIST2, the proteins listed in Table 1 and Akt signalingproteins. In embodiments, small molecule inhibitors against PI3K/Aktsignaling include, but are not limited to, Wortmannin, demethoxyviridin,LY294002, perifosine, idelalisib, buparlisib (BKM 120), duvelisib,alpelisib (BYL719), TGR 1202, copanlisib (BAY 80-6946), PX-866,dactolisib, RP6530, SF1126, INK1117, pictilisib, XL147 (also known asSAR245408), XL765 (also known as SAR245409), palomid 529, GSK1059615,ZSTK474, PWT33597, CUDC-907, ME-401, IPI-549, IC87114, TG100-115,CAL263, RP6503, PI-103, GNE-477, and AEZS-136.

In embodiments, TWIST signaling inhibitor used in any method describedherein is within a composition described herein. In embodiments, TWISTsignaling inhibitor used in any method described herein is within apharmaceutical composition described herein.

According to the methods provided herein, cancer is any type of cancer.In embodiments, the cancer to be treated is melanoma, ovarian cancer,breast cancer, prostate cancer, lung cancer, glioblastoma multiforme,neuroblastoma, kidney cancer and more. In embodiments, the cancer to betreated is a metastatic cancer. In embodiments, the cancer to be treatedis metastatic melanoma, metastatic ovarian cancer, metastatic breastcancer, metastatic prostate cancer, metastatic lung cancer, metastaticglioblastoma multiforme, metastatic neuroblastoma, metastatic kidneycancer and more.

The terms effective amount and effective dosage are usedinterchangeably. The term effective amount is defined as any amountnecessary to produce a desired physiologic response. In this case, forexample, a desired physiologic response includes a subject being more(e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%or more) responsive to the anti-cancer agent when administered with aTWIST signaling inhibitor compared to the response level of the subjectwithout taking the TWIST signaling inhibitor. A skilled artisan wouldreadily determine the signs of being responsive (such as, slowerprogression of cancer, smaller size of the cancer tissue, etc.).Effective amounts and schedules for administering the agent may bedetermined empirically, and making such determinations is within theskill in the art. The dosage ranges for administration are those largeenough to produce the desired effect in which one or more symptoms ofthe disease or disorder are affected (e.g., reduced or delayed). Thedosage should not be so large as to cause substantial adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sex,type of disease, the extent of the disease or disorder, route ofadministration, or whether other drugs are included in the regimen, andcan be determined by one of skill in the art. The dosage can be adjustedby the individual physician in the event of any contraindications.Dosages can vary, and can be administered in one or more doseadministrations daily, for one or several days. Guidance can be found inthe literature for appropriate dosages for given classes ofpharmaceutical products.

A therapeutically effective amount used herein also refers to an amountof TWIST signaling inhibitor that is sufficient to re-sensitize thesubject to a subsequent or co-treatment with an anti-cancer agent. Inembodiments, the amount of the TWIST signaling inhibitor used herein issufficient to make the subject become responsive to the anti-canceragent that is administered simultaneously or subsequently or inalteration.

In embodiments, the TWIST signaling inhibitor and the anti-cancer agentare administered in a single composition. In embodiments, the TWISTsignaling inhibitor and the anti-cancer agent are both bound to a samedelivery vehicle (e.g., a nanoparticle).

In embodiments, the TWIST signaling inhibitor and the anti-cancer agentare administered in two or more compositions. In embodiments, the TWISTsignaling inhibitor and the anti-cancer agent are bound to differentdelivery vehicles (e.g., different nanoparticles).

In some embodiments, the release of the anti-cancer agent is controlledby nanovalves of the nanoparticle. In particular, either an internal oran external stimulus (such as lower pH, magnetic field or light)triggers the opening of the nanovalves, thus controlling the release ofthe anti-cancer drug.

In embodiments, the TWIST signaling inhibitor and the anti-cancer agentare administered in a single composition, as being bound to the samedelivery vehicle (e.g., a nanoparticle). In embodiments, the TWISTsignaling inhibitor and the anti-cancer agent are releasedsimultaneously. In embodiments, the anti-cancer agent is released afterthe TWIST signaling inhibitor takes effect. In embodiments, theanti-cancer agent is released one or more hours, or one or more daysafter the TWIST signaling inhibitor takes effect. In embodiments, theanti-cancer drug is released about 30 min, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, 30 days or more after the TWIST signaling inhibitor takeseffect.

In some embodiments, the TWIST signaling inhibitor and the anti-canceragent are administered in two or more compositions, as being bound todifferent delivery vehicles (e.g., different nanoparticles).

In embodiments, the compositions containing TWIST signaling inhibitorand the anti-cancer agent are administered simultaneously. Inembodiments, the anti-cancer agent is released simultaneously as theTWIST signaling inhibitor takes effect. In embodiments, the anti-canceragent is released about 30 min, 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, Iday, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29days, 30 days or more after the TWIST signaling inhibitor takes effect.

In embodiments, the composition(s) containing the anti-cancer agent isadministered after the administration of the composition containing theTWIST signaling inhibitor. In embodiments, the composition(s) containingthe anti-cancer agent is administered and released about 30 min, 1 hour,2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26days, 27 days, 28 days, 29 days, 30 days or more after the TWISTsignaling inhibitor is administered and takes effect.

The TWIST signaling inhibitor takes effect when the expression level ofTWIST signaling gene or TWIST signaling protein or the activity level ofTWIST signaling protein is less than 90% of the initial level, less than80% of the initial level, less than 70% of the initial level, less than60% of the initial level, less than 50% of the initial level, less than40% of the initial level, less than 30% of the initial level, less than20% of the initial level or less than 10% of the initial level. Methodsfor determining the expression level of TWIST signaling gene or TWISTsignaling protein or the activity level of TWIST signaling protein iswell known in the art.

In embodiments, the nanoparticle utilized in these methods are furtherbound with one or more tumor targeting moieties (such as HA) to enhancethe tumor-targeting specificity of the nanoparticle.

EMBODIMENTS

Embodiments contemplated herein include embodiments P1 to P41 following.

Embodiment P1

A composition comprising an anti-TWIST siRNA bound to a nanoparticle.

Embodiment P2

The composition of embodiment P1, wherein said anti-TWIST siRNAcomprises a sequence of any one of SEQ ID Nos: 1-10, or a complementarysequence thereof.

Embodiment P3

The composition of embodiment P2, wherein said sequence comprises anucleic acid modification.

Embodiment P4

The composition of embodiment P3, wherein said nucleic acid modificationis a 2′-O-methyluracil or inverted abasic deoxyribose.

Embodiment P5

The composition of embodiment P4, wherein said anti-TWIST siRNAcomprises a sequence of SEQ ID NO: 11.

Embodiment P6

The composition of embodiment P3, wherein said modification is a2-thio-deoxyuracil.

Embodiment P7

The composition of embodiment P6, wherein said anti-TWIST siRNAcomprises a sequence of SEQ ID NO: 12.

Embodiment P8

The composition of embodiment P1, wherein said nanoparticle is amesoporous silica nanoparticle (MSN).

Embodiment P9

The composition of embodiment P8, wherein said MSN is bound topolyethyleneimine (PEI).

Embodiment P10

The composition of embodiment P9, wherein said MSN is further bound to atumor targeting moiety.

Embodiment P11

The composition of embodiment P10, wherein said tumor targeting moietyis hyarulonic acid (HA).

Embodiment P12

The composition of embodiment P8, wherein said MSN is bound to a tumortargeting moiety.

Embodiment P13

The composition of embodiment P12, wherein said tumor targeting moietyis HA.

Embodiment P14

The composition of embodiment P8, wherein said MSN comprises a low pHactivated nanovalve.

Embodiment P15

The composition of embodiment P14, wherein said MSN is further boundwith an anti-cancer agent.

Embodiment P16

The composition of embodiment P8, wherein said MSN is a magneticcore-shell MSN.

Embodiment P17

The composition of embodiment P16, wherein said MSN is further boundwith an anti-cancer agent.

Embodiment P18

The composition of embodiment P1, wherein said nanoparticle is adendrimer-based nanoparticle.

Embodiment P19

The composition of embodiment P18, wherein said dendrimer-basednanoparticle is YTX3-15.

Embodiment P20

The composition of embodiment P19, wherein said anti-cancer agent isDoxorubicin, Cisplatin, Carboplatin, Taxanes, Camptothecin or anycombination thereof.

Embodiment P21

The composition of embodiment P1, further comprising a pharmaceuticallyacceptable excipient to form a pharmaceutical composition.

Embodiment P22

An siRNA comprising a sequence of any one of SEQ ID NOs: 1-12.

Embodiment P23

A DNA sequence encoding an siRNA sequence comprising a sequence of anyone of SEQ ID Nos: 1-10.

Embodiment P24

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient and said siRNA of embodiment P22.

Embodiment P25

A method of reversing resistance to an anti-cancer drug in a subject,the method comprising administering an effective amount of a TWISTinhibitor to said subject.

Embodiment P26

The method of embodiment P25, wherein said TWIST inhibitor is a TWIST1inhibitor.

Embodiment P27

The method of embodiment P25, wherein said TWIST inhibitor is ananti-TWIST siRNA.

Embodiment P28

The method of embodiment P25, wherein said TWIST inhibitor is thecomposition of one of embodiments P1 to P24.

Embodiment P29

A method of treating cancer in a subject in need thereof, the methodcomprising administering to said subject a therapeutically effectiveamount of a TWIST inhibitor.

Embodiment P30

The method of embodiment P29, wherein said TWIST inhibitor is ananti-TWIST siRNA.

Embodiment P31

The method of embodiment P29, wherein said TWIST inhibitor is thecomposition of one of embodiments P1 to P24.

Embodiment P32

The method of embodiment P29, wherein said therapeutically effectiveamount is an amount sufficient to sensitize the subject to subsequenttreatment with an anti-cancer agent.

Embodiment P33

The method of embodiment P29, further comprising administering to saidsubject a therapeutically effective amount of said anti-cancer agent.

Embodiment P34

The method of embodiment P33, wherein said anti-cancer agent isDoxorubicin, Cisplatin, Carboplatin, Taxanes, Camptothecin or anycombination thereof.

Embodiment P35

The method of embodiment P33, wherein said anti-cancer agent is bound toa nanoparticle.

Embodiment P36

The method of embodiment P35, wherein said nanoparticle is MSN.

Embodiment P37

The method of embodiment P36, wherein said MSN comprises a low pHactivated nanovalve or is a magnetic core-shell MSN.

Embodiment P38

The method of embodiment P29, wherein said subject is resistant to saidanti-cancer agent.

Embodiment P39

A method of inhibiting metastasis in a subject in need thereof, themethod comprising administering to said subject a therapeuticallyeffective amount of a TWIST inhibitor.

Embodiment P40

The method of embodiment P39, wherein said TWIST inhibitor is ananti-TWIST siRNA.

Embodiment P41

The method of embodiment P39, wherein said TWIST inhibitor is thecomposition of one of embodiments P1 to P24.

Embodiment P42

The method of embodiment P39, further comprising administering to saidsubject a therapeutically effective amount of an anti-cancer agent.

Embodiment P43

The method of embodiment P42, wherein said anti-cancer agent is bound toa nanoparticle.

Further embodiments include embodiments Q1 to Q19 following.

Embodiment Q1

A method of treating cancer in a subject in need thereof, the methodcomprising administering to said subject a therapeutically effectiveamount of a TWIST signaling inhibitor.

Embodiment Q2

A method of inhibiting metastasis in a subject in need thereof, themethod comprising administering to said subject a therapeuticallyeffective amount of a TWIST signaling inhibitor.

Embodiment Q3

A method of reversing resistance to an anti-cancer drug in a subject,the method comprising administering to said subject a therapeuticallyeffective amount of a TWIST signaling inhibitor.

Embodiment Q4

The method of any one of embodiments Q1 to Q3, wherein said TWISTsignaling inhibitor is an inhibitor of growth arrest-specific 6 (GAS6),L1 cell adhesion molecule (L1CAM) or an Akt signaling factor.

Embodiment Q5

The method of embodiment Q4, wherein said Akt signaling factor isphosphatidylinositol 3-kinase (PI3K) or protein kinase B (Akt).

Embodiment Q6

The method of any one of embodiments Q1 to Q5, wherein said TWISTsignaling inhibitor is a siRNA inhibitor or a small molecule inhibitor.

Embodiment Q7

The method of any one of embodiments Q1 to Q6, wherein said TWISTsignaling inhibitor is bound to a delivery vehicle.

Embodiment 18

The method of embodiment 7, wherein said delivery vehicle is ananoparticle or a lipid particle.

Embodiment Q9

The method of embodiment Q1 or embodiment Q2, wherein said subject isresistant to an anti-cancer drug.

Embodiment Q10. The method of embodiment Q1 or embodiment Q2, whereinsaid therapeutically effective amount is an amount sufficient tore-sensitize the subject to subsequent treatment with an anti-canceragent.

Embodiment Q11

The method of any one of embodiments Q1 to Q10, further comprisingadministering to said subject a therapeutically effective amount of ananti-cancer agent.

Embodiment Q12

The method of any one of embodiments Q1 to Q11, wherein said anti-canceragent is doxorubicin, cisplatin, carboplatin, a taxane, camptothecin orany combination thereof.

Embodiment Q13

The method of any one of embodiments Q1 to Q12, wherein said inhibitoris within a pharmaceutical composition comprising said inhibitor and apharmaceutically acceptable excipient.

Embodiment Q14

A composition comprising a TWIST signaling inhibitor bound to a deliveryvehicle.

Embodiment Q15

The composition of embodiment Q14, wherein said delivery vehicle is ananoparticle or a lipid particle.

Embodiment Q16

The composition of embodiment Q14, wherein said TWIST signalinginhibitor is an inhibitor of growth arrest-specific 6 (GAS6), L1 celladhesion molecule (L1CAM) or an Akt signaling factor.

Embodiment Q17

The composition of embodiment Q16, wherein said Akt signaling factor isphosphatidylinositol 3-kinase (PI3K) or protein kinase B (Akt).

Embodiment Q18

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient and a composition of embodiment Q14.

Embodiment Q19

A kit, comprising an instruction manual and a composition of embodimentQ14 or a pharmaceutical composition of embodiment Q18.

Further embodiments contemplated herein include embodiments 1 to 55following.

Embodiment 1

A composition comprising a TWIST signaling inhibitor bound to a deliveryvehicle.

Embodiment 2

The composition of embodiment 1, wherein said TWIST signaling inhibitoris an siRNA inhibitor or a small molecule inhibitor.

Embodiment 3

The composition of embodiment 1, wherein said delivery vehicle is ananoparticle or a lipid particle.

Embodiment 4

The composition of embodiment 2, wherein said siRNA inhibitor is ananti-TWIST siRNA.

Embodiment 5

The composition of embodiment 4, wherein said anti-TWIST siRNA comprisesa sequence of any one of SEQ ID Nos: 1-10, or a complementary sequencethereof.

Embodiment 6

The composition of embodiment 5, wherein said sequence comprises anucleic acid modification.

Embodiment 7

The composition of embodiment 6, wherein said nucleic acid modificationis a 2′-O-methyluracil or inverted abasic deoxyribose.

Embodiment 8

The composition of embodiment 7, wherein said anti-TWIST siRNA comprisesa sequence of SEQ ID NO: 11.

Embodiment 9

The composition of embodiment 6, wherein said modification is a2-thio-deoxyuracil.

Embodiment 10

The composition of embodiment 9, wherein said anti-TWIST siRNA comprisesa sequence of SEQ ID NO: 12.

Embodiment 11

The composition of embodiment 3, wherein said nanoparticle is amesoporous silica nanoparticle (MSN).

Embodiment 12

The composition of embodiment 11, wherein said MSN is bound topolyethyleneimine (PEI).

Embodiment 13

The composition of embodiment 12, wherein said MSN is further bound to atumor targeting moiety.

Embodiment 14

The composition of embodiment 13, wherein said tumor targeting moiety ishyarulonic acid (HA).

Embodiment 15

The composition of embodiment 11, wherein said MSN is bound to a tumortargeting moiety.

Embodiment 16

The composition of embodiment 15, wherein said tumor targeting moiety isHA.

Embodiment 17

The composition of embodiment 11, wherein said MSN comprises a low pHactivated nanovalve.

Embodiment 18

The composition of embodiment 17, wherein said MSN is further bound withan anti-cancer agent.

Embodiment 19

The composition of embodiment 11, wherein said MSN is a magneticcore-shell MSN.

Embodiment 20

The composition of embodiment 19, wherein said MSN is further bound withan anti-cancer agent.

Embodiment 21

The composition of embodiment 3, wherein said nanoparticle is adendrimer-based nanoparticle.

Embodiment 22

The composition of embodiment 21, wherein said dendrimer-basednanoparticle is YTX3-15.

Embodiment 23

The composition of embodiment 20, wherein said anti-cancer agent isdoxorubicin, cisplatin, carboplatin, a taxane, camptothecin or anycombination thereof.

Embodiment 24

The composition of embodiment 1, wherein said TWIST signaling inhibitoris an inhibitor of growth arrest-specific 6 (GAS6), L1 cell adhesionmolecule (L1CAM) or an Akt signaling factor.

Embodiment 25

The composition of embodiment 24, wherein said Akt signaling factor isphosphatidylinositol 3-kinase (PI3K) or protein kinase B (Akt).

Embodiment 26

The composition of embodiment 1, further comprising a pharmaceuticallyacceptable excipient to form a pharmaceutical composition.

Embodiment 27

An siRNA comprising a sequence of any one of SEQ ID NOs: 1-12.

Embodiment 28

A DNA sequence encoding an siRNA sequence comprising a sequence of anyone of SEQ ID Nos: 1-10.

Embodiment 29

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient and said siRNA of embodiment 27.

Embodiment 30

A method of reversing resistance to an anti-cancer drug in a subject,the method comprising administering an effective amount of a TWISTsignaling inhibitor to said subject.

Embodiment 31

The method of embodiment 30, wherein said TWIST signaling inhibitor is aTWIST1 inhibitor.

Embodiment 32

The method of embodiment 30, wherein said TWIST signaling inhibitor isan anti-TWIST siRNA.

Embodiment 33

The method of embodiment 30, wherein said TWIST signaling inhibitor isan inhibitor of growth arrest-specific 6 (GAS6), L1 cell adhesionmolecule (L1CAM) or an Akt signaling factor.

Embodiment 34

The method of embodiment 33, wherein said Akt signaling factor isphosphatidylinositol 3-kinase (PI3K) or protein kinase B (Akt).

Embodiment 35

The method of embodiment 30, wherein said TWIST signaling inhibitor isthe composition of embodiment 1.

Embodiment 36

A method of treating cancer in a subject in need thereof, the methodcomprising administering to said subject a therapeutically effectiveamount of a TWIST signaling inhibitor.

Embodiment 37

The method of embodiment 36, wherein said TWIST signaling inhibitor isan anti-TWIST siRNA.

Embodiment 38

The method of embodiment 36, wherein said TWIST signaling inhibitor isan inhibitor of growth arrest-specific 6 (GAS6), L1 cell adhesionmolecule (L1CAM) or an Akt signaling factor.

Embodiment 39

The method of embodiment 38, wherein said Akt signaling factor isphosphatidylinositol 3-kinase (PI3K) or protein kinase B (Akt).

Embodiment 40

The method of embodiment 36, wherein said TWIST signaling inhibitor isthe composition of embodiment 1.

Embodiment 41

The method of embodiment 36, wherein said therapeutically effectiveamount is an amount sufficient to sensitize the subject to subsequenttreatment with an anti-cancer agent.

Embodiment 42

The method of embodiment 36, further comprising administering to saidsubject a therapeutically effective amount of said anti-cancer agent.

Embodiment 43

The method of embodiment 42, wherein said anti-cancer agent isdoxorubicin, cisplatin, carboplatin, a taxane, camptothecin or anycombination thereof.

Embodiment 44

The method of embodiment 42, wherein said anti-cancer agent is bound toa nanoparticle.

Embodiment 45

The method of embodiment 44, wherein said nanoparticle is MSN.

Embodiment 46

The method of embodiment 45, wherein said MSN comprises a low pHactivated nanovalve or is a magnetic core-shell MSN.

Embodiment 47

The method of embodiment 36, wherein said subject is resistant to ananti-cancer agent.

Embodiment 48

A method of inhibiting metastasis in a subject in need thereof, themethod comprising administering to said subject a therapeuticallyeffective amount of a TWIST signaling inhibitor.

Embodiment 49

The method of embodiment 48, wherein said TWIST signaling inhibitor isan anti-TWIST siRNA

Embodiment 50

The method of embodiment 48, wherein said TWIST signaling inhibitor isan inhibitor of growth arrest-specific 6 (GAS6), L1 cell adhesionmolecule (L1CAM) or an Akt signaling factor.

Embodiment 51

The method of embodiment 50, wherein said Akt signaling factor isphosphatidylinositol 3-kinase (PI3K) or protein kinase B (Akt).

Embodiment 52

The method of embodiment 48, wherein said TWIST signaling inhibitor isthe composition of embodiment 1.

Embodiment 53

The method of embodiment 48, further comprising administering to saidsubject a therapeutically effective amount of an anti-cancer agent.

Embodiment 54

The method of embodiment 53, wherein said anti-cancer agent is bound toa nanoparticle.

Embodiment 55

A kit, comprising an instruction manual and a composition of embodiment1 or a pharmaceutical composition of embodiment 29.

EXAMPLES Example 1—Mesoporous Silica Nanoparticle Delivery of ChemicallyModified siRNA Against TWIST1 Leads to Reduced Tumor Burden

Abstract. Growth and progression of solid tumors depends on theintegration of multiple pro-growth and survival signals, including theinduction of angiogenesis. TWIST1 is a transcription factor whosereactivation in tumors leads to epithelial to mesenchymal transition(EMT), including increased cancer cell stemness, survival, andinvasiveness. Additionally, TWIST1 drives angiogenesis via activation ofIL-8 and CCL2, independent of VEGF signaling. In this work, resultssuggest that chemically modified siRNA against TWIST1 reverses EMT bothin vitro and in vivo. siRNA delivery with a polyethyleneimine-coatedmesoporous silica nanoparticle (MSN) led to reduction of TWIST1 targetgenes and migratory potential in vitro. In mice bearing xenografttumors, weekly intravenous injections of the siRNA-nanoparticlecomplexes resulted in decreased tumor burden together with a loss ofCCL2 suggesting a possible anti-angiogenic response. Therapeutic use ofTWIST1 siRNA delivered via MSNs has the potential to inhibit tumorgrowth and progression in many solid tumor types.

In this disclosure we demonstrate siRNA delivery via a(polyethyleneimine) PEI coated MSN. We show that the siRNA is able toknock down the expression of TWIST1 both in vitro and in vivo followingMSN delivery. The knockdown of TWIST1 resulted in a functional change inthe expression of TWIST1 targets and ultimately leads to decreased tumorburden in a xenograft mouse model.

Methods.

MSN Production. The 100 nm MSNs used were produced using the sol-gelmethod as described previously.[33] The addition of the cationic PEIcoating to the MSNs has previously been described.[40]

Cell Culture and Transfection. MDA-MB-435S melanoma cancer cells wereobtained from ATCC (Manassas, Va.). These cells were maintained at 37°C., 5% CO₂, and 90% humidity in a standard tissue culture incubator.Media for the MDA-MB-435S cells consisted of RPMI 1640 media (GeneseeScientific, San Diego, Calif.) supplemented with 10% fetal bovine serumand 1% penicillin/streptomycin. Cells were passaged using 0.25% trypsin(Genesee Scientific, San Diego, Calif.) every 3-4 days as they becameconfluent.

To allow for imaging of cells in a xenograft model, it was necessary tocreate a stable line of MDA-MB-435S that expressed GFP and fireflyluciferase (ffluc). These cells were created by with the aid of a CMVlentiviral construct that encodes a fusion protein of GFP and fflucseparated by a three glycine linker.[41] This stable cell line wasmaintained and used for all experiments (in vitro and in vivo).

Previously published siRNA sequences against TWIST1 were used(si419-passenger, 5′-GGACAAGCUGAGCAAGAUU-3′(SEQ ID NO: 1); si419-guide,5′-AAUCUUGCUCAGCUUGUCCUU-3′(SEQ ID NO: 2); si494-passenger,5′-GCGACGAGCUGGACUCCAA-3′(SEQ ID No: 3); si494-guide,5′-UUGGAGUCCAGCUCGUCGCUU-3′(SEQ ID No: 4)).[42] Two chemicalmodifications (addition of 2′-O-methyl and inverted abasic reoxyribose,see FIG. 1D) were made to the si419 passenger/sense strand for allexperiments except for those involving LIPOFECTAMINE® 2000 transfection.The chemically modified si419 duplex is referred to as si419Hybrid orsi419H. No chemical modifications were made to the si494 sequences.siRNA duplexes were formed by placing equal molar volumes together andheating them in a hot block (100° C.) for 10 minutes followed by removalof the aluminum block from the heat source. Block and duplexes were thenallowed to cool to room temperature over several hours. The negativecontrol siRNA (siQ, labeled with ALEXAFLUOR® 647) was AllStars NegativeControl siRNA from Qiagen (Valencia, Calif.; proprietary sequence).

Transient transfection of MDA-MB-435S GFP+ffluc was carried out usingLIPOFECTAMINE® 2000 (Thermo Fisher Scientific, Waltham, Mass.) accordingto the manufacturer's instructions. LIPOFECTAMINE® 2000 transfection wasused to confirm the functionality of the siRNA (si419 and si494) priorto testing their efficacy with MSNs. Transfection of siRNA with MSNs wascarried out by incubating the MSNs with the siRNA overnight at 4° C.while rotating the tube constantly. The mixture consisted of 7 parts MSN(diluted to 500 ng/ul in sterile PBS) to 1 part siRNA (diluted to 10uM). The final concentration for of MSNs and siRNA applied to cells was17.5 ng/ul and 50 nM, respectively.

ELISA Assay. Expression of Interleukin 8 (IL-8) is known to be mediatedby TWIST1. Therefore, we used an ELISA assay to measure the amount ofIL-8 secreted by MDA-MB-435S GFP+ffluc cells following treatment withMSN+siRNA (si419H and si494). MSNs complexed with siRNA against GFP(siGFP) was used as a control. In a 6-well tissue culture plate, 250,000MDA-MB-435S GFP+ffluc cells were seeded and allowed to adhere for 24hours. Following this acclimatization period, cells were incubated withthe MSN+siRNA complexes for 72 hours at standard tissue cultureconditions. After 72 hours, a sample of the conditioned media wascollected for secreted IL-8 quantification. The IL-8 Human ELISA Kit(Thermo Fisher Scientific Inc., Waltham, Mass.) was used according tothe manufacturer's specifications.

Western Blot. Following siRNA treatment described above, cells werelifted from tissue culture wells with 0.25% trypsin, pelleted, and lysedin RIPA buffer. Protein concentration was determined using a BCA Assay(Thermo Fisher Scientific). A total of 30 μg of protein per lane was runon 4% stacking and 12% resolving polyacrylamide gels. Following gelelectrophoresis, protein was transferred to Immobilon-P PVDF membrane(Millipore, Billerica, Mass.) using a Trans-Blot SD Semi-Dry TransferCell (Bio-Rad, Hercules, Calif.). Membranes were then blocked with 5%dry milk dissolved in 1×PBS with 0.1% Tween-20. Antibodies were dilutedin blocking buffer (1:250 for anti-TWIST1 and 1:2,500 for anti-Actin).Antibodies used were: anti-TWIST, TWIST 2c1a (Santa Cruz Biotech,Dallas, Tex.); anti-j3-Actin, A1978 (Sigma Aldrich, St. Louis, Mo.); andHorse Radish Peroxidase (HRP)-conjugated anti-mouse secondary antibody(Li-Cor, Lincoln, Nebr.). ECL Plus chemiluminescent substrate (Pierce,Thermo Fisher Scientific, Waltham, Mass.) and Blue Devil Film (GeneseeScientific, San Diego, Calif.) were used for the development of images.

Wound Healing Assay. In vitro wound healing assays were performed toexamine directional cell migration.[43] MDA-MB-435S GFP+ffluc cells weregrown in the tissue culture conditions described previously in 6-welltissue culture plates. Cells were treated with MSN+siQ, MSN+si419H, orMSN+si494 for 24 hours (as described earlier in this section) prior tobeing scratched. A sterile 200l pipette tip was used with consistentpressure to scratch a line in the monolayer of cells. Images were takenat several time points thereafter using a Nikon TE-2000S microscope(Nikon, Tokyo, Japan) and SPOT Advanced software (DiagnosticInstruments, Sterling Heights, Mich.). Markings were made on the lid ofthe tissue culture plate to ensure that the same location along thescratch was imaged at each time point. Cells were incubated withMSN+siRNA complexes at 37° C., 5% CO₂, and 90% humidity in a tissueculture incubator at all times except for the imaging time points.

Quantitative PCR. Total cellular RNA was isolated using the RNeasy Pluskit (Qiagen, Valencia, Calif.). Synthesis of cDNA from total RNA wascarried out using the iScript cDNA Synthesis kit (Bio-Rad, Hercules,Calif.) with an equal amount of RNA used for all samples. QuantitativeRT-PCR was performed using Maxima SYBR Green Master Mix (Thermo FisherScientific, Waltham, Mass.) in 25l reactions. Thermocycling wasconducted in a Bio-Rad iQ5 thermal cycler for 40 cycles (95° C., 15s;57° C., 60s; 79° C., 30s) followed by melt curve analysis. Data wereanalyzed using Bio-Rad iQ5 software. Primers used were: TWIST1 forward,5′-CTATGTGGCTCACGAGCGGCTC-3′(SEQ ID No: 14); TWIST1 reverse,5′-CCAGCTCCAGAGTCTCTAGACTGTCC-3′ (SEQ ID No: 15); Vimentin forward,5′-TCGTCACCTTCGTGAATACCAAGA-3′ (SEQ ID NO: 16), Vimentin reverse,5′-CCTCAGGTrCAGGGAGGAAAAGTT-3′(SEQ ID NO: 17); CCL2 forward,5′-CAGCCAGATGCAATCAATGCC-3′(SEQ ID NO: 18); CCL2 reverse,5′-TGGAATCCTGAACCCACTTCT-3′ (SEQ ID NO: 19).[18].

Confocal Microscopy. MDA-MB-435S GFP+ffluc cells were seeded into a 3.5cm glass bottom tissue culture dish and allowed to attach over a 24 hourperiod. At that time, 2 ml of fresh media was added following theremoval of old media. Next, MSN+siQ complexes (labeled with ALEXAFLUOR®647) were added to the dish for a 24 hour period at the finalconcentrations of 17.5 ng/μl (MSN) and 50 μM (siQ). Following fixationwith 4% paraformaldehyde cells were counterstained with DAPI (300 nM for2 min) and then mounted using PROLONG® Gold (Thermo Fisher Scientific,Waltham, Mass.). Confocal images were obtained using the Zeiss LSM 700Confocal Microscope and ZEN 2012 microscopy software (Zeiss A G,Oberkochen, Germany).

Tumor Engraftment and In vivo Imaging. All animal work was donefollowing protocol approval by the Institutional Animal Care and UseCommittee of the City of Hope Beckman Research Institute. A total of 18female NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ (NSG) mice (The JacksonLaboratory, Bar Harbor, Me.) were used. All mice were approximately 10weeks old at the time of the inoculation of tumor cells. Mice wererandomly divided into four groups: control mice (no xenografts, 2 mice);negative control (MSN+siQ, 4 mice); si419H treatment group (MSN+si419H,6 mice); and si494 treatment group (MSN+si494, 6 mice). Mice (other thanthe no xenograft controls) received bilateral inoculations into the4^(th) mammary fat pad set immediately adjacent to the nipple.Inoculation was carried out while mice were fully anesthetized usingisoflurane (2-5%) delivered via a vaporizer. Inoculum for each mammaryfat pad consisted of 3.2×10⁶ MDA-MB-435S GFP+ffluc cells suspended in 75μl PBS. Following injections, mice were allowed to fully recover in aclean cage before being placed back in their home cage.

Bioluminescent imaging of mice began 12 days after initial inoculationof tumor cells and occurred every week for six weeks. Images werecaptured using the Xenogen IVIS 100 biophotonic imaging system (STTARR,Toronto, Ontario, Canada) in order to follow xenograft growth. Prior tobeing fully anesthetized with isoflurane (2-4%), mice were given a 200μl intraperitoneal injection of 25 mg/ml D-Luciferin (PerkinElmer,Waltham, Mass.). Ten minutes after the D-luciferin injection, mice wereplaced in a black box inside of the biophotonic imager. Images werecaptured over a period of one minute.

Intravenous (IV) injections of MSN+siRNA (siQ, si419H, or si494; siQfluorescently labeled with ALEXAFLUOR® 647, si419H and si494 labeledwith Cy5) were started two weeks after the inoculation of MDA-MB-435SGFP+ffluc cells and done weekly for six weeks. Prior to IV injections,mice were briefly warmed with a heat lamp and then placed in arestrainer. A 120 μl volume of MSN+siRNA was given in the lateral tailvein of each mouse (excluding no-tumor controls). The injectionconsisted of 105 μl of 500 ng/μl MSN complexed with 15 μl of 10 μM siRNA(complexing took place overnight at 4° C.). Ten minutes after the IVinjection animals were fully anesthetized (2-4% isoflurane) andunderwent infrared imaging using the Xenogen IVIS 100 biophotonicimaging system (STTARR, Toronto, Ontario, Canada).

At the end of the experiments, all animals were euthanized via CO₂asphyxiation followed by a complete necropsy. Tumors were carefullydissected away from any adherent tissue and weighed and then placed in10% formalin along with the heart, lungs, spleen, kidney, and liver forhistological evaluation. Histopathological examination of tissues wasinterpreted by a board certified veterinary pathologist who was blindedto the treatment groups.

Statistical Analysis. Data were analyzed using Prism 6 (GraphPadSoftware, La Jolla, Calif.). All qPCR data were analyzed by a one-tailedunpaired t-test with Welch's correction, separately comparing si419H tosiQ and si494 to siQ. As data were analyzed as mean, standard deviation,and n, no correction for multiple comparisons was performed. ELISA datawere analyzed by Kruskal-Wallis non-parametric test and Dunn's test formultiple comparisons. Data represents normalized data from two runs,each in duplicate, for a total n of 4. Tumor growth data were analyzedby Kruskal-Wallis and Dunn's test as described above, and represent allsingle tumors in each group (grown as 2 tumors per mouse). Exact pvalues are given on figures where applicable (*=p<0.05 and **=p<0.01).

SUM 1315 Cell Culture. SUM 1315 breast cancer cells were obtained fromATCC (Manassas, Va.). Tissue culture incubator environment wasmaintained at 37° C., 5% CO₂, with 90% humidity. Cells were grown inmedia consisting of a 50-50 mixture of DMEM and F12 media, supplementedwith 5% fetal bovine serum, 10 ng/ml EGF, 5 g/ml insulin, and 1%penicillin/streptomycin. Upon reaching confluency cells were passagedevery 2-3 days using 0.25% trypsin (Genesee Scientific, San Diego,Calif.).

Stable SUM1315 cell lines were developed that reliably expressed a shorthairpin RNA (shRNA) against TWIST1 (shTwist419, shTwist494), or ascrambled control shRNA (shScram) as a negative control. These SUM1315cell lines were created as previously described. See e.g, Li S, et al.,BMC Biology. 2012; 10: 73

RNA-Seq. Cell pellets from SUM1315-shTwist419, SUM1315-shTwist494, andSUM1315-shScram were collected and immediately processed for RNAextraction. Total cellular RNA was isolated using the RNeasy Plus kit(Qiagen, Valencia, Calif.). 10 μg of total RNA was then resuspended innuclease free water and poly (A) enriched to remove ribosomal RNA.Samples were processed using the Illumina HiSeq 2500 (Illumina, SanDiego, Calif.). Resulting raw RNA-seq data was first aligned usingTopHat (version 2.0.8, Center for Computational Biology, Johns HopkinsUniversity) followed by counting and expression scoring with Cufflinks(version 2.02) as described previously. See e.g., Trapnell C, et al.Nature protocols. 2012; 7: 562-78.

Immunohistochemistry. To confirm that nests of cells in lung fields weremetastatic lesions 5 μm sections of all paraffin imbedded tissues werecut and stained with 1:3000 rabbit polyclonal GFP antibodies (Ab290,Abcam, Cambridge, Mass.). Sections were incubated at room temperaturefor 30 minutes. An HRP-conjugated goat anti-rabbit IgG was used as thesecondary antibody.

MTT Assay. The tetrazolium dye MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was usedto assess cell death following treatment with various concentrations ofMSN+siRNA. A total of 5,000 MDA-MB-435S GFP+ffluc were placed in eachwell of a 96-well tissue culture plate and allowed to attach over 24hours. Next the cells were treated with either ½×, 1×, 2×, 5×, or 10×the typical MSN+siRNA concentration (final concentration for of MSNs andsiRNA applied to cells was 17.5 ng/ul and 50 nM, respectively) for 24hours. Following this incubation period media was removed from each welland 110 μl MTT diluted in complete media (0.45 mg/ml) was added.Incubation lasted for 3 hours at 37° C., 5% CO₂, with 90% humidity.Following the incubation period the MT media was removed and 110 μl DMSOwas added to each well and the plate was gentle shaken for 15 mins. Dyeintensity for each well was then read at a wavelength of 580 nm.

Results.

siRNA design and loading onto mesoporous silica nanoparticles. We havedesigned and synthesized siRNA to inhibit TWIST expression, byincorporating various chemical modifications to increase resistance tonuclease activity, decrease immunogenicity and to promote efficientloading of the guide strand into RISC (FIG. 1C). Our previous studieswith a breast cancer cell line (SUM 1315) demonstrated the efficacy ofthe si419H and si494.[42] To confirm efficacy of si419 and si494(with/without chemical modifications) in MDA-MB-435S cells,Lipofectamine® 2000 transfection was performed. A time dependent TWIST1knockdown, was observed with greater than 90% TWIST1 protein reductionat 72 hours following transfection (FIG. 2A). Chemical modifications(2′-O-methyl and inverted abasic reoxyribose on passenger/sense strand)did not impact the efficacy of TWIST1 knock down (FIG. 6).

The above siRNAs were loaded onto mesoporous silica nanoparticles (MSNs)that have a PEI coated cationic surface. Mixing of PEI+siRNA enablestight binding of siRNA to positively charged MSNs. Thus, PEI providesprotection of siRNA and efficient delivery of siRNA to cancer cells.

MSN+siRNA delivery and TWIST1 silencing. A stable line of MDA-MB-435Sthat expresses GFP and ffluc was successfully produced via lentiviraltransduction. Both GFP and ffluc were shown to be fully functional invitro and in vivo (FIGS. 7A-7C). Confocal microscopy of MDA-MB-435SGFP+ffluc cells following incubation with fluorescently labeled MSN+siQresulted in correct perinuclear localization of the siRNA (FIG. 2B). Nonoticeable cellular death was observed. MTT assays confirmed thatsignificant cell death did not occur until MSN+siRNA treatment was 2-5times the usual concentration (i.e. MSNs=17.5 ng/μl and siRNA=50 nM)(FIG. 5).

TWIST1 knock down in MDA-MB-435S GFP+ffluc cells was observed at 72hours post MSN+siRNA treatment. A 90% decrease in TWIST1 was observed inboth RNA and protein measurements (FIG. 2C), whereas basal levels ofTWIST1 expression returned by one week.

TWIST knockdown results in decreased migration and decrease in thesecretion of IL8. TWIST1 knock down resulted in downstream effects tofunctional phenotypes following MSN+siRNA treatment. A wound healingassay showed an appreciable difference in the migration capabilities ofMDA MB 435S GFP+ffMuc cells following treatment with MSN+si419H andMSN+si494 when compared to the MSN+siQ control (FIG. 3A). IL-8 ELISAassays also demonstrated significant reduction in human IL-8 secretionfrom the MDA-MB-435S GFP+ffMuc cells following 72 hours of treatmentwith MSN+si419H or MSN+494 when compared to MSN+siQ negative control(FIG. 3B). Reduction of IL-8 secretion was also observed at 48 and 96hours post-transfection, however, no IL-8 changes observed at 24 hours.

Tumor Burden Decreased Following Treatment with MSN+Chemically ModifiedsiRNA. All mice that received an inoculation of tumor cells developedbilateral tumors in the area of the mammary fat pad in the 4^(h) mammaryset. All tumors were palpable and emitted a robust bioluminescent signalfollowing IP injection of D-Luciferin. Following histopathologicalexamination, no changes were observed in the heart, lungs, liver,spleen, and kidneys, in mice receiving MSN+siRNA treatments whencompared to controls that did not receive any treatment.

However, tumors collected from the MSN+si419H mice were significantlysmaller when compared to the weights of the tumors from the MSN+siQcontrol mice (FIGS. 4A-4B). Furthermore, by visual inspection, the bloodvessels supplying the tumors of MSN+si419H treated mice were smaller andappeared less hemorrhagic than those of the other two groups (FIG. 4A).However, the tumors from mice treated with MSN+si494 without chemicalmodifications were not significantly smaller than tumors of the controlmice as would be expected from the in vitro TWIST1 knockdown studies forboth si419H and si494 in (FIG. 2C).

Tumor Characterization Demonstrated EMT Inhibition. mRNA isolated fromcollected tumors were analyzed for relative quantities of TWIST1,Vimentin (an EMT marker), and CCL2 (chemokine involved in angiogenesis).A significant reduction in the amount of TWIST1, Vimentin, and CCL2 mRNAwas observed in the MSN+si419H and MSN+si494 treated mice when comparedto the control mice (MSN+siQ) (FIGS. 4C-4E). The average relativereduction of TWIST1 for MSN+si494 treated mice was less than that ofthose treated with MSN+si419H (p=0.0067), though no significantdifference was observed for Vimentin or CCL2. Thus, these data suggestinhibition of EMT/angiogenesis through the delivery of TWIST1 siRNA.

No Evidence of Decreased Metastatic Lesions. Contrary to what would beexpected with significant TWIST1 knock down, there was no reduction inthe number of metastatic lung lesions (FIGS. 8A-8C). Metastatic lesionswere categorized into 4 groups based on size, and no significantdifference was seen among the MSN+siQ, MSN+si419H, and MSN+si494treatment groups. The cause for this finding could be attributed to twopossible elements in the experimental design. First, the initial MSNtreatment occurred two weeks after the MDA-MB-435S GFP+ffluc wereinoculated. This period of time was designed to allow for the tumorcells to engraft unencumbered by any treatment. However, it is possiblethat the tumor cells from this highly metastatic cell line spread to thelungs and engrafted before MSN treatments were initiated. Although lessclinically relevant, future studies with this cell line might benefitfrom beginning MSN+siRNA treatments simultaneously with tumor cellinoculation. Second, just because the in vitro data demonstrated TWIST1protein levels following MSN+siRNA treatment were reduced at 72 hours,but returned by 7 days (FIG. 2C); it is possible that in vivo levels ofTWIST1 (and its target genes) are higher and for effective knocked downit would have been necessary to provide MSN+siRNA treatments at morefrequent intervals (e.g. twice per week rather than once). Metastaticdisease is the ultimate cause of mortality in the vast majority ofcancer related deaths. For this reason there are great efforts beingmade in understanding the mechanisms underlying metastasis as well asdeveloping therapies to exploit these mechanisms to prevent the spreadof cancer cells.^(44, 45)

Discussion

Here, we demonstrate the effective delivery of a chemically modifiedsiRNA therapy via a silica nanoparticle carrier. Following delivery ofthe siRNA, there is significant knockdown of the transcription factorTWIST1, a known regulator of EMT and angiogenesis.[46] TWIST1 knockdownwas associated with decreased tumor burden in vivo as well as areduction in the TWIST1-mediated targets—Vimentin (EMT) and CCL2(angiogenesis) (FIGS. 4C-4E).

Our results demonstrate that nanoparticle delivery of TWIST1 siRNA leadsto a decrease in tumor burden supporting the idea that TWIST1 is animportant therapeutic target. TWIST1 was selected as a target for siRNAtherapy because it is highly associated with metastasis, EMT, and a poorprognosis.[46] TWIST1 is also an attractive therapeutic target becauseit is not expressed in most adult tissues, and therefore most normaltissues would not be negatively impacted by an siRNA silencingstrategy.[47]

The observed decrease of tumor burden appears to be due to the effect ofreduced TWIST expression on EMT-mediated angiogenesis. Angiogenesis incancer occurs following a variety of complex signaling pathways thatultimately result in increased blood supply to the tumor, thus allowingfor continued growth and metastasis. Two key components of angiogenesisthat were examined here are CCL2 and IL-8. CCL2 is a monocytechemotactic protein secreted by tumor cells responsible for recruitingmacrophages to aid in establishing new blood vessels in thetumor.[18,49] IL-8 is a pro-inflammatory cytokine that is known to worksynergistically with VEGF to stimulate vessel growth in tumors.[50]Furthermore, IL-8 is known to promote angiogenesis independent of VEGFand can cause anti-VEGF therapies to fail.[51] Reduction of both IL-8(in vitro, secreted) and CCL2 (in vivo) were observed followingtreatment with MSN+si419H and MSN+si494 (FIGS. 3B and 4E). Whilereduction of these two promoters of angiogenesis was evident for bothtypes of MSN+siTWIST treatment groups, reduced tumor burden was onlyobserved with MSN+si419H.

In our experiments, we found that si419H exhibited excellent efficacy invitro and in vivo.

Our results show that mesoporous silica nanoparticles provide efficientvehicle delivery of siRNA in vitro and in vivo. The MSNs developed forthis project were shown in vitro to successfully deliver their siRNApayload into melanoma cells (FIG. 2B) and that this delivery resulted insignificant knockdown of TWIST1 (FIG. 2C). These results furtherestablish that MSNs are viable carriers for siRNA.[36,54] The PEI coated130 nm MSNs used in this project were shown to cause no cellular deathin vitro (at normal concentrations, FIG. 5). Following weekly IVinjections of MSN+siRNA there was no observable histopathologic evidenceof tissue damage in any of the examined organ tissues. Taken together,this would indicate that MSNs are efficacious and safe as reportedpreviously.[55,56] MSNs provide a number of advantages for futuredevelopment as a siRNA vehicle. First, MSNs are highly customizable inboth their size and shape, thus allowing for a finely tuned nanoparticlewhich can be optimized to specific delivery needs. Modification to thesize and structure of the MSN allows for increased biocompatibility andsafety.[55] The porous nature of the MSN allows for the internal loadingof therapies in addition to the siRNA demonstrated here and elsewhere,thus enabling a multimodal approach to cancer therapy.[54] Finally, MSNscan be modified to carry targeting moieties that allow them to home tospecific tissues or tumors.[57] The porous nature of the MSNs used inthis research is an untapped nanoparticle characteristic that should beexplored in conjunction with the observed knockdown of TWIST1. Drugresistance is a major hindrance to the treatment of cancer and oftenresults in a more aggressive phenotype. Therefore, a MSN basedco-delivery strategy of anti-TWIST1 siRNA together with chemotherapycould result in more pronounced tumor reduction. Increased efficacy andreduced dosage would also be possible with tumor targeting moieties aspreviously described.[54]

These data support continued development and optimization of MSNs as adelivery platform for the treatment of cancer. This research representsthe first example of silencing an EMT regulating transcription factorfollowing siRNA delivery using MSNs.

References (Example 1)

[1] Nguyen D X, et al., Nature reviews Cancer. 2009; 9: 274-84; [2]Coghlin C. & Murray G I, J Pathol. 2010; 222: 1-15; [3] Lee J M, et al.,J Cell Biol. 2006; 172: 973-81; [4] Kalluri R. & Weinberg R A, J ClinInvest. 2009; 119: 1420-8; [5] Vernon A E & LaBonne C., Curr Biol. 2004;14: R719-21; [6] Niu R F, et al., J Exp Clin Cancer Res. 2007; 26:385-94; [7] Carmeliet P., Nature. 2005; 438: 932-6; [8] Risau W.,Nature. 1997; 386: 671-4; [9] Pralhad T, et al., The Journal of pharmacyand pharmacology. 2003; 55: 1045-53; [10] Carmeliet P. & Jain R K,Nature. 2000; 407: 249-57; [11] Hanahan D. & Weinberg R A, Cell. 2011;144: 646-74; [12] Spano D. & Zollo M., Clin Exp Metastasis. 2012; 29:381-95; [13] Gotink K J & Verheul H M, Angiogenesis. 2010; 13: 1-14;[14] Choi S H, et al., Liver international: official journal of theInternational Association for the [Study of the Liver. 2014; 34: 632-42;[15] Semenza G L, Journal of cellular biochemistry. 2007; 102: 840-7;[16] Yancopoulos G D, et al., Nature. 2000; 407: 242-8; [17] Linardou H,et al., Breast Cancer Res. 2012; 14: R145; [18] Low-Marchelli J M, etal., Cancer Res. 2013; 73: 662-71; [19] Bialek P, et al., Dev Cell.2004; 6: 423-35; [20] Khan M A, et al., Tumour biology: the journal ofthe International Society for Oncodevelopmental Biology and Medicine.2013; 34: 2497-506; [21] El Ghouzzi V, et al., Eur J Hum Genet. 1999; 7:27-33; [22] Xu Y, et al., Am J Pathol. 2013; 183: 1281-92; [23] Kong D,et al., Cancers (Basel). 2011; 3: 716-29; [24] Vesuna F, et al.,Neoplasia. 2009; 11: 1318-28; [25] Li J, et al., Investigativeophthalmology & visual science. 2014; 55: 8267-77; [26] Shalini SinghIWYM, et al., Advances in Biology. 2014; 2014: 8 Pages; [27] MironchikY, et al., Cancer Res. 2005; 65: 10801-9; [28] Li S, et al., BMC Biol.2012; 10: 73; [29] Lu J, et al., Small. 2007; 3: 1341-6; [30] Liong M,et al., ACS nano. 2008; 2: 889-96; [31] Greish K., Methods Mol Biol.2010; 624: 25-37; [32] Castanotto D. & Rossi J J, Nature. 2009; 457:426-33; [33] Hom C., et al., Small. 2010; 6: 1185-90; [34] David S., etal., Pharmacol Res. 2010; 62: 100-14; [35] Yamazaki Y., et al., Genetherapy. 2000; 7: 1148-55; [36] Xia T, et al., ACS nano. 2009; 3:3273-86; [37] Guo P, et al., Adv Drug Deliv Rev. 2010; 62: 650-66; [38]Behlke M A, Oligonucleotides. 2008; 18: 305-19; [39] Czauderna F, etal., Nucleic Acids Res. 2003; 31: 2705-16; [40] Meng H, et al., ACSnano. 2011; 5: 4131-44; [41] Brown C E, et al., Cancer Res. 2009; 69:8886-93; [42] Finlay J., et al., BioMed research international. 2015;2015: 382745; [43] Liang C C, et al., Nature protocols. 2007; 2: 329-33;[44] Vernon A E, et al., Rev Endocr Metab Disord. 2007; 8: 199-213; [45]Kuperwasser C., et al., Cancer Res. 2005; 65: 6130-8; [46] Yang J., etal., Cell. 2004; 117: 927-39; [47] Wang S M, et al., Gene. 1997; 187:83-92; [48] Dykxhoorn D M & Lieberman J., Cell. 2006; 126: 231-5; [49]Bonapace L., et al., Nature. 2014; 515: 130-3; [50] Martin D., et al., JBiol Chem. 2009; 284: 6038-42; [51] Huang D., et al., Cancer Res. 2010;70: 1063-71; [52] Teng Y. & Li X., Clin Exp Metastasis. 2014; 31:367-77; [53] Forsbach A, et al., J Immunol. 2008; 180: 3729-38; [54]Meng H, et al., ACS nano. 2013; 7: 994-1005; [55] Lu J., et al., Small.2010; 6: 1794-805; [56] Ferris D P, et al., Small. 2011; 7: 1816-26;[57] Tarn D. et al., Accounts of chemical research. 2013; 46: 792-801;[58] Vesuna F., et al., Oncogene. 2011; [59] Wang X, et al., Oncogene.2004; 23: 474-82; [60] Yanes R E & Tamanoi F., Therapeutic delivery.2012; 3: 389-404;

Example 2—RNA-Based TWIST Inhibition Via Dendrimer Complex to ReduceBreast Cancer Cell Metastasis

Abstract.

Breast cancer is the leading cause of cancer-related deaths among womenin the United States, and five-year survival rates for patients withmetastases are drastically lower than for patients with localizeddisease. This is especially true for patients with triple-negativebreast cancer (TNBC; ER, PR, Her2 negative) tumors. Understanding themetastatic mechanisms of aberrant cancer cells is therefore crucial toidentify new therapeutic targets and develop novel therapies to use inconjunction with current therapies to prevent metastasis and improvepatient outcomes. A potential target is the TWIST1 transcription factor,a master regulator of cellular migration through epithelial-mesenchymaltransition (EMT), which is often overexpressed in aggressive breastcancers. Here, we demonstrate an siRNA-based TWIST1 silencing approachwith delivery using a modified poly(amidoamine) (PAMAM) dendrimer. Ourresults demonstrate that SUM 1315 TNBC cells efficiently take upPAMAM-siRNA complexes, leading to significant knock down of TWIST1 andEMT-related target genes. Knockdown lasts up to one week posttransfection and leads to a reduction in the migratory and invasivenature of breast cancer cells, as determined by in vitro wound healingand transwell assays. Furthermore, we demonstrate that PAMAM dendrimerscan deliver siRNA to xenograft orthotopic tumors with a high degree ofspecificity. These results suggest that dendrimer-based delivery ofsiRNA for TWIST1 silencing may be a valuable adjunctive therapy forpatients with TNBC.

In the current study, we investigated whether anti-TWIST1 siRNA could befunctionally delivered to metastatic breast cancer cells (SUM 1315cells) using YTZ3-15. We tested the ability of the YTZ3-15-deliveredsiRNA to knock down TWIST1, reduce expression of EMT-related targetgenes, and alter the phenotypic characteristics associated with cancercell migration (metastasis). We also evaluated the tumor-specificdelivery capability of YTZ3-15 using a mouse breast cancer model.

Materials and Methods.

Cell Culture, Transfection, and Stable Cell Line Production. SUM 1315breast cancer cells were obtained from ATCC (Manassas, Va.). Cells weremaintained at 37° C., 5% CO₂, and 90% humidity in a tissue cultureincubator. Media for SUM 1315 cells consisted of a 50-50 mixture of DMEMand F12 media, supplemented with 5 μg/ml insulin, 10 ng/ml EGF, 5% fetalbovine serum, and 1% penicillin/streptomycin. Cells were passaged using0.25% trypsin (Genesee Scientific, San Diego, Calif.) every 2-3 days asthey reached confluency. Transient transfection of SUM 1315 cells wasperformed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham,Mass.) or YTZ3-15 (obtained from Dr. Ling Peng, CentreInterdisciplinaire de Nanoscience de Marseille, France). siRNA sequenceswere as follows: siTwistA-sense, 5′-GGACAAGCUGAGCAAGAUU-3′(SEQ ID NO:1); siTwistA-antisense, 5′-AAUCUUGCUCAGCUUGUCCUU-3′(SEQ ID NO:2);siTwistB-sense, 5′-GCGACGAGCUGGACUCCAA-3′(SEQ ID No: 3);siTwistB-antisense, 5′-UUGGAGUCCAGCUCGUCGCUU-3′(SEQ ID No: 4). Allcustom siRNAs were synthesized by IDT (Integrated DNA Technologies,Inc., Skokie, Ill.) and arrived lyophilized and were resuspened in H2Oprior to being reannealed. Negative control siRNA (siQ, labeled witheither AlexaFluor® 488 or 647) was AllStars Negative Control siRNA fromQiagen (Valencia, Calif.). Lipofectamine 2000 was diluted fifty-fold inOptiMEM® (Thermo Fisher Scientific; Waltham, Mass.) and incubated with10 uM siRNA for 20 min at room temperature. YTZ3-15 was diluted to 1.75uM in Opti-MEM® and mixed with 10 uM siRNA at an N/P ratio of 5, for afinal dendrimer-siRNA complex concentration of 50 nM. Complexes wereincubated 20 min at room temperature which resulted in dendriplexes ofroughly 100 nm in size. Incubation of the YTZ3-15 dendriplexes with SUM1315 cells was done at the tissue culture conditions describedpreviously for up to 7 days with fresh media being added to existingmedia as needed. Stable transfections of SUM 1315 cells were performedusing lentivirus. Cells expressing eGFP-firefly luciferase fusionprotein were created by transfecting the CMV cassette as describedpreviously [39].

To examine the effects of TWIST1 knock down in SUM 1315 cells withoutthe possible confounding variables of the delivery mechanism itself, wedeveloped cell lines that stably expressed a short hairpin RNA (shRNA)against TWIST1 (shTwistA, shTwistB), or a scrambled control shRNA(shScram) as a negative control. Cells were alternatively stablytransfected with shTwistA, shTwistB or shScram as described previously[40]. Immortalized human mesenchymal stem cells (hMSCs) were used asdescribed previously [41].

Wound Healing Assay. To examine direction cell migration in vitro woundhealing assays were performed as described previously [42]. In summary,SUM 1315 cells (parental, eGFP+luc, shScram, shTwistA, and shTwistB)were grown in the conditions described above (2.1) in 6-well tissueculture plates. Once cells reached 80% confluency, a sterile 200 μlpipette tip was used to scratch a line in the monolayer of cells. Imageswere taken immediately after the scratch and at several time pointsthereafter using a Nikon TE-2000S microscope and SPOT Advanced software(Diagnostic Instruments, Sterling Heights, Mich.). Care was taken toalways capture images in the same location for each time point.Additionally, SUM 1315 cells were transfected with siQ, siTwistA, orsiTwistB using YTZ3-15 at a 50 nM final concentration. Cells wereincubated with YTZ3-15+siRNA for 24 hours at 37° C., 5% CO₂, and 90%humidity in a tissue culture incubator, the plates were then scratchedwith the 200 μl pipette tip.

Invasion Assay. 2.5×10⁵ SUM 1315 cells were transfected using YTZ3-15complexed with siQ, siTwistA, or siTwistB as described above (2.2).After 24 hours incubation, 2.5×10⁵ SUM 1315 cells were lifted from theplate with 0.25% trypsin, washed, and seeded onto transwell inserts (8μm pore diameter Millipore, Darmstadt, Germany). Inserts (which nestinside of wells in a 24-well tissue culture plate) were pre-coated withMatrigel® (3 mg/ml, 60 μl, diluted with serum-free medium) (BDBiosciences, San Jose, Calif.), which was allowed to solidify in atissue culture incubator for 30 minutes prior to the addition of thetransfected cells. In order to stimulate cell invasion the top chambercontaining the transfected SUM 1315 cells contained 1% FBS (400 μl)while the lower chamber contained 600 μl of complete media with 20% FBS.Cells were permitted to invade for 24 hours in a tissue cultureincubator. After this period the MatriGel® and any remaining cells inthe upper chamber were removed with a cotton-tipped swab. Transwellmembranes were then washed twice in PBS and stained with Crystal Violet.Five images of each membrane were taken and cells were counted manually.Graphs were created to show an average count from each of the fiveimages.

Quantitative PCR. Total cellular RNA was isolated using the RNeasy Pluskit (Qiagen, Valencia, Calif.). RNA quantity and quality was measuredand analyzed (with 260/280 nm and 260/230 nm spectra measurements) usinga NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, Mass.). An equalamount of RNA for all conditions was used as a template for cDNAsynthesis using the iScript cDNA Synthesis kit (Bio-Rad, Hercules,Calif.). Quantitative RT-PCR was performed in quadruplicate using MaximaSYBR Green Master Mix (Thermo Fisher Scientific, Waltham, Mass.) in 25lreactions. Cycling was conducted in a Bio-Rad iQ5 thermal cycler for 40cycles (95° C., 15s; 57° C., 60s; 79° C., 30s) followed by melt curveanalysis. Data were analyzed using Bio-Rad iQ5 software using the2^(−ΔΔ) ^(Ct) method and normalized to Actin. Primers used were: Twistforward #1, 5′-CTATGTGGCTCACGAGCGGCTC-3′(SEQ ID No: 14); Twist reverse#1, 5′-CCAGCTCCAGAGTCTCTAGACTGTCC-3′(SEQ ID No: 15); Twist forward #2,5′-TCTTACGAGGAGCTGCAGACGCA-3′(SEQ ID NO: 20); Twist reverse #2,5′-ATCTTGGAGTCCAGCTCGTCGCT-3′(SEQ ID NO: 21); N-cadherin forward,5′-GGGACAGTTCCTGAGGGATCAA-3′(SEQ ID NO: 22), N-cadherin reverse,5′-TGGAGCCTGAGACACGATrCTG-3′(SEQ ID NO: 23), Vimentin forward,5′-TCGTCACCTTCGTGAATACCAAGA-3′(SEQ ID NO: 16), Vimentin reverse,5′-CCTCAGGTrCAGGGAGGAAAAGTT-3′(SEQ ID NO: 17), 3 Actin forward,5′-CCGCAAAGACCTGTACGCCAAC-3′ (SEQ ID NO: 24); 3 Actin reverse,5′-CCAGGGCAGTGATCTCCTTCTG-3′ (SEQ ID NO: 25).

Western Blot. Cells were seeded at 250,000 cells per well in 6-welltissue culture plates and treated as described in section 2.1. Cellswere pelleted, lysed in RIPA buffer, and protein concentration wasdetermined using the BCA Assay (Thermo Fisher Scientific, Waltham,Mass.). 30 μg total protein per lane was run on 4% stacking and 10-12%resolving polyacrylamide gels and transferred to Immobilon-P PVDFmembrane (Millipore, Billerica, Mass.). Membranes were blocked with 5%dry milk dissolved in 1×PBS with 0.1% Tween-20. Antibodies were dilutedin blocking buffer. Antibodies used were: anti-Twist, Twist 2c1a (SantaCruz Biotech, Dallas, Tex.); anti-β-actin, A1978 (Sigma Aldrich, St.Louis, Mo.); and HRP-conjugated anti-mouse secondary antibodies. ECLPlus chemiluminescent substrate (Pierce, Thermo Fisher Scientific,Waltham, Mass.) and Blue Devil Film (Genesee Scientific, San Diego,Calif.) were used.

Confocal Microscopy. SUM 1315 cells were transfected with siQ siRNA(labeled with AlexaFluor® 647) using YTZ3-15 and incubated for 24 hoursin a tissue culture incubator. Cells were then treated with LysoTracker®Red (Thermo Fisher Scientific, Waltham, Mass.) according to themanufacturer's conditions. Confocal images were obtained using the ZeissLSM 700 Confocal Microscope and the ZEN 2012 microscopy software (ZeissA G, Oberkochen, Germany).

Tumor Engraftment. A total of nine female NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, Me.)were used to engraft the SUM 1315 eGFP+luc breast cancer cells (8 weeksold at time of inoculation). The nine mice were to be divided into threegroups; intratumoral (IT), intravenous (IV), and IV long term. Whileunder anesthesia (Isoflurane, 2.5-4%), mice received bilateralinoculations of cells into the 4^(h) mammary fat pad. Inoculum for eachmammary fat pad consisted of 1×10⁶ SUM 1315 eGFP+luc cells together with2×10⁵ hMSCs resuspended in 50 μl Matrigel®. Injections were deliveredinto the mammary fat pad adjacent to the nipple. Mice were then allowedto recover in a clean cage. Two NSG mice receiving no cells were used ascontrols.

In Vivo Imaging. After tumor cell inoculations, the mice were imagedevery two weeks using the Xenogen IVIS 100 biophotonic imaging system(STTARR, Toronto, Ontario, Canada) to monitor tumor growth. To obtain invivo images, mice were given a 200 μl intraperitoneal (IP) injection of25 mg/ml D-Luciferin (PerkinElmer, Waltham, Mass.). After a 10-minutewaiting period, animals were anesthetized using Isoflurane (2-5%) andplaced in a black box in the biophotonic imager. Bioluminescent imageswere captured over a period of one minute. Once tumors had reached0.5-0.75 cm, three mice were given a single intravenous (IV) injectionof the YTZ3-15+siQ complex diluted in 200 μl PBS. A separate group ofthree animals was given intratumoral injections of the YTZ3-15+siQcomplex. Three additional tumor bearing animals received IV injectionsof YTZ3-15+siQ to be used for longer term imaging studies with the finalimages being captured 14 days post injection. After the injections,animals underwent in vivo fluorescent imaging using the biophotonicimager (Cy5.5 filter). Images were captured at 5, 10, 15, and 240minutes after injection of the dendrimer complex. After the final timepoint (4 hours), all animals were euthanized and tissues were collected.Tumors, spleen, kidney, and liver from each animal were imaged ex vivousing the IVIS 100 to detect the AlexaFluor® 647-labeled siRNA withoutthe hindrance of the skin and fur. Two NSG mice receiving no cells wereused as controls for in vivo imaging.

Statistics and Replications. Wound healing assays were repeated threetimes as were the western blot analyses. Invasion assay was repeatedtwice with identical conditions. Five images were captured for eachinvasion assay condition and the numbers of cells were counted manuallyand standard deviations were calculated using Excel (Microsoft, Redman,Wash.). Quantitative PCR experiments were done in quadruplicate andanalyzed using the 2^(−ΔΔ) ^(Ct) method in the Bio-Rad iQ5 software.Three animals per group (along with two control animals) were used forbiodistribution purposes and no statistical analyses were performed within vivo imaging data.

Results and Discussion.

Stable TWIST1 Knock Down in SUM 1315 Cells. The relationship betweenTWIST1 expression and EMT has been established for breast cancer [43].To examine the effects of TWIST1 knock down in SUM 1315 cells withoutthe possible confounding variables that a delivery mechanism may cause,we developed cell lines that stably expressed a shRNA against TWIST1(shTwistA and shTwistB) and a scrambled shRNA (shScram) as a negativecontrol. TWIST1 expression in the SUM 1315 shTwistA and shTwistB celllines demonstrated excellent knock down of TWIST1 compared to theparental line and the shScram line (FIGS. 11A-11B).

To test the effect of TWIST1 knock down on cell movement, we performed awound healing assay. Our results demonstrated that the SUM 1315 shTwistAand shTwistB cell lines had reduced directional migratory abilitiescompared to the SUM 1315 shScram cell line (FIG. 11C). Taken together,these data suggest that shTwistA and shTwistB not only significantlyknock down expression of TWIST1 in SUM 1315 cells, but also that thedown regulation of TWIST1 results in a phenotypic change consistent withdiminished migratory ability.

siRNA-Mediated TWIST1 Knock Down in SUM 1315 Cells. The SUM 1315 shRNAresults described above not only demonstrate a significant reduction inthe amount of TWIST1 expression, but also a phenotypic change in cellmigration, suggesting that these shRNA sequences were effective inknocking down TWIST1 expression. We thus designed siRNA sequences(siTwistA and siTwistB) based on these shRNA sequences. To test theefficacy of siTwistA and siTwistB, SUM 1315 cells were transfected usingLIPOFECTAMINE® 2000. Transfection with both siTwistA and siTwistBresulted in knockdown of TWIST1, with siTwistB giving slightly moreknockdown than siTwistA in both protein and mRNA levels. Next, we testedthe delivery of siRNA into SUM 1315 cells using the YTZ3-15 dendrimer.Cellular uptake efficiency of AlexaFluor® 647 labeled siQ (acting as asurrogate for unlabeled siTwistA and siTwistB) was greater than 90%after 24 hours, as measured with flow cytometry and fluorescentmicroscopy (FIGS. 12A-12B). The presence of siQ transfected usingYTZ3-15 was confirmed as far out as 7 days from the time of transfection(FIG. 12B). These findings confirm previous work [38] performed withthis PAMAM dendrimer and demonstrate its ability to safely deliver siRNAacross the cell membrane because we did not appreciate any increase incell death. Cellular uptake efficiency using YTZ3-15+siQ was comparablewhen tested in other cell lines including other breast, ovarian,uterine, and prostate cancer cell lines (data not shown).

While uptake of the dendrimer complex can be appreciated withfluorescent microscopy and flow cytometry, these methods do not indicatethe location of the siRNA within the cell. To examine this, we usedLysoTracker® Red (dye taken up into acidic organelles) to show where siQis colocalized. Our results show that much of the siQ signal colocalizeswith the mid to late endosome in the SUM 1315 eGFP+luc cell line (FIG.12C). This colocalization is desirable to take advantage of the “protonsponge effect”, which is thought to be essential for siRNA release[44,45].

After confirming the function of siTwistA and siTwistB withLipofectamine 2000 and the cellular uptake efficiency of siQ usingYTZ3-15, we tested siTwistA and siTwistB with YTZ3-15-based delivery.TWIST1 levels were measured using qPCR and found to be significantlyreduced at 24 hours and one week post transfection (FIG. 13A). TwoEMT-related TWIST1 target genes (Vimentin and N-Cadherin) also showedreduced mRNA expression. Vimentin and N-Cadherin were both substantiallyreduced at the 24 hour time point; however, Vimentin showed a slightreturn at the one week time point whereas N-Cadherin continued todecrease at one week (FIG. 13B). While reduced expression of these geneswas noted, renewed expression of the epithelial marker E-Cadherin wasnot observed (data not shown). This is a noted difference from previousstudies [4]. The possible causes for this discrepancy are the differentcell lines used between ours and previous studies, and that E-Cadherinis not entirely controlled by TWIST1 [19,46]. Reduced expression ofthese EMT-related genes is a positive indication that migration andinvasion would be hindered.

Next, we performed a wound healing assay to validate thatYTZ3-15-delivered siTwistA and siTwistB not only reduces TWIST1 and itstarget genes, but also inhibits the migratory action of SUM 1315 cells.This assay demonstrated decreased directional migration of SUM 1315cells transfected with siTwistA (FIG. 13C).

The EMT process consists of migration and invasion, and TWIST1 is amajor player in allowing cancer cells to infiltrate surrounding tissues,blood vessels and the lymphatic system [40,47]. To investigate whetherthe invasive phenotype is reduced following siRNA-mediated TWIST1 knockdown, we performed a transwell invasion assay. Results indicate that theYTZ3-15+siRNA-treated cells have diminished abilities to invade theMATRIGEL® Matrix and pass through the porous membrane, thus indicating areduction in the invasive phenotype (FIG. 13D). TWIST1 overexpression isassociated with cancers that are more metastatic and therefore invasive[22], and these data show that TWIST1 silencing following PAMAMdendrimer delivery of siRNA decreases metastatic potential. This in turnsuggests that as a therapeutic approach for patients with MBC, thisdelivery method and target could have a significant impact on improvingsurvival and outcomes for MBC patients if pre-clinical and clinicaltrials show similar results.

A TWIST1 siRNA therapeutic approach to assist in the treatment of MBC isalso attractive because it could complement and augment currenttreatment regimens. The data suggest that siRNA-based knock down ofTWIST1 could be used in conjunction with hormonal therapy orchemotherapy to achieve a synergistic effect.

In vivo Distribution of PAMAM Dendrimers. In vivo studies were completedto determine the optimum delivery route (IV versus IT) of siQ usingYTZ3-15. Five minutes after the IV and IT injection of the YTZ3-15+siQcomplex, a bright signal was noted at the site of the tumor (FIG. 14A).The signal at the tumor site continued to be evident in mice thatreceived IT injections at 10, 15, and 240 minutes, whereas no signal wasseen at the tumor site after 5 minutes in mice that received IVinjections (FIG. 14A). Ex vivo imaging of tumors, spleen, liver, andkidneys revealed a robust ALEXAFLUOR® 647 signal in the tumors butlittle to no signal in other examined organs (FIG. 14B). This ex vivotumor-centric signal was evident for all mice, regardless of the routeof administration (IV versus IT).

While the YTZ3-15 dendrimer does not have any inherent tumor-targetingcapabilities, our in vivo studies demonstrate that this PAMAM dendrimerdoes accumulate preferentially in the orthotopic breast cancer tumors.It is possible that localization to the tumor is due to the EnhancedPermeability and Rentention (EPR) effect, which has been seen with otherPAMAM dendrimers and nanoparticle delivery vehicles [58-60]. Theinherent leakiness of tumor vasculature coupled with minimal lymphaticdrainage results in particles becoming trapped and consequentlyconcentrated in the tumor environment. This effect is magnified as thetumor enlarges and promotes angiogenesis, which may explain why siQconcentration was noted only after orthotopic tumors reached 0.5×0.5 cmin size (data not shown).

Conclusions

These studies demonstrate successful delivery and utilization of twosiRNAs against TWIST1. Delivery was realized using a modified thirdgeneration PAMAM dendrimer and resulted in significant knock down ofTWIST1 and other EMT-related target genes. TWIST1 knock down resulted ina reduction in cellular migration and invasion as has been observedpreviously [9,11,40,47,61]. Finally, delivery of an siRNA by YTZ3-15 wasshown to have a specific concentrating ability in orthotopic tumors in atriple negative (ER-negative, progesterone receptor [PR]-negative andHER2-negative) breast cancer mouse model.

These data demonstrated that TWIST1 is an important and potentiallyclinically significant therapeutic target for the treatment of MBC aswell as other solid tumor cancers [62-64]. These data furtherdemonstrated that TWIST1 knock down via PAMAM dendrimer-delivered siRNAcould serve as a valuable tool and adjuvant therapy to reducemigration/invasion, chemoresistance, and anti-apoptotic tendencies.Novel results from this study serve to validate a multimodal approach tocancer treatment by focusing on a transcription factor associated withbreast cancer tumor types that have minimal treatment options.Furthermore, these data demonstrated (both in vitro and in vivo) the useof siRNA coupled with nanoparticles to treat malignant breast cancer byknocking down Twist 1 and its associated EMT targets.

References (Example 2)

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Example 3—Nanoparticle Delivery of siRNA Against TWIST to Reduce DrugResistance and Tumor Growth in Ovarian Cancer Models

Abstract. Epithelial ovarian cancer (EOC) is the most deadly gynecologicmalignancy on account of its late stage at diagnosis and frequency ofdrug resistant recurrences. Novel therapies to overcome these barriersare urgently needed. TWIST is a developmental transcription factorreactivated in cancers and linked to angiogenesis, metastasis, cancerstem cell phenotype, and drug resistance, making it a promisingtherapeutic target. In this work, we demonstrate the efficacy of TWISTsiRNA (siTWIST) and two nanoparticle delivery platforms to reversechemoresistance in EOC models. Polyamidoamine dendrimers and mesoporoussilica nanoparticles (MSNs) carried siTWIST into target cells and led tosustained TWIST knockdown in vitro. Mice treated with cisplatin plusMSN-siTWIST exhibited lower tumor burden than mice treated withcisplatin alone, with most of the effect coming from reduction indisseminated tumors. This platform has potential application forovercoming the clinical challenges of metastasis and chemoresistance inEOC and other TWIST overexpressing cancers.

Background.

Epithelial ovarian cancer (EOC) is one of the leading causes ofcancer-related deaths among women worldwide, representing a significantunmet therapeutic challenge [1-3]. EOC accounts for 90% of all ovariancancers [1]; additionally, 70% of women with EOC are not diagnosed untilthe disease is advanced stage [1]. The majority of EOC patients respondwell to first line chemotherapy consisting of a platinum drug and/orpaclitaxel. Unfortunately, most of these patients relapse with diseasethat is both metastatic and drug resistant, with a five-year survivalrate of approximately 20% [3-5]. There is therefore an urgent need fortherapies to prevent both metastatic spread and acquired drug resistancein EOC.

EOC tumors are characterized by high expression levels ofepithelial-to-mesenchymal transition (EMT) markers such as TWIST, whichplays an essential role in cancer metastasis (FIG. 15A) [6,7]. However,transcription factors such as TWIST are difficult to target with smallmolecule drugs due to their nuclear localization [16]. To circumventthis issue, small interfering RNAs (siRNAs) have become increasinglypopular. We have designed and validated two therapeutic siRNAs againstTWIST (FIG. 15C), and have evaluated two nanoparticle-based deliveryplatforms. First, we used third generation polyamidoamine (PAMAM)dendrimers, and second, we created polyethylenimine (PEI) coatedmesoporous silica nanoparticles (MSNs). We have previously showneffective delivery to tumor cells using both of these modalities, bothin vitro and in mouse models of melanoma and breast cancer [14,15].

In this study we applied our siRNA-nanoparticle technologies to EOC. Wehypothesized that nanoparticle delivered anti-TWIST siRNAs would knockdown TWIST and sensitize cells to chemotherapeutics. We also evaluatedthe tumor-specific delivery capability of our MSNs. By evaluating theeffects of TWIST knockdown using MSNs in animal models of a metastaticand chemoresistant phenotype, we present an MSN delivery platform forsiRNA and drug combination therapies to prevent both metastatic spreadand acquired drug resistance in ovarian and other cancers.

Methods.

Cell Culture. A2780R and all derivatives of Ovcar8 were grown in RPMI1640 (Genesee Scientific, San Diego, Calif.) in a tissue cultureincubator at 37° C., 5% CO₂, and 90% humidity. Growth medium wassupplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.Cells were passaged every 2-4 days using 0.25% trypsin (GeneseeScientific). Where indicated, cells were transfected with Lipofectamine2000 (Thermo Fisher, Waltham, Mass.) according to the manufacturer'sinstructions. A2780R cells are a cisplatin resistant derivative ofA2780.

Optimization of Ovcar8 for in vivo use. For mouse experiments, Ovcar8cells were stably transfected with CMV-p:EGFP-ffluc pHIV7 as has beendescribed previously [17]. This resulted in expression of aneGFP-firefly luciferase (ffluc) fusion protein. To increase engraftmentefficiency and homogeneity of the cell population, Ovcar8 cells werepassaged through mice. Ovcar8-GFP+ffluc cells were injectedintraperitoneally (IP) and allowed to form tumors. Cells were harvestedafter 37 days and used to establish the Ovcar8-IP line.

siRNA design. siRNAs against TWIST were designed based on shRNAs thatwere previously validated [13,15]. Sequences are: si419 guide,5′-AAUCUUGCUCAGCUUGUCCUU-3′ (SEQ ID NO:2); si419 passenger,5′-GGACAAGCUGAGCAAGAUU-3′ (SEQ ID NO:1); si494 guide,5′-UUGGAGUCCAGCUCGUCGCUU-3′ (SEQ ID NO:4); si494 passenger,5′-GCGACGAGCUGGACUCCAA-3′ (SEQ ID NO:3). Non-targeting control siRNA(siQ) was AllStars Negative Control siRNA, labeled with AlexaFluor-647,from Qiagen (Valencia, Calif.). For in vivo studies, 2′-O-methyluraciland inverted abasic ribose chemical modifications were made to the si419passenger strand, as illustrated in FIG. 18A [14].

PAMAM dendrimer design and delivery. The polyamidoamine (PAMAM)dendrimer used in this study is the YTZ3-5 third generation dendrimer,illustrated in FIG. 16A and previously described [15,18]. To complex thedendrimer with siRNA, YTZ3-15 at 240 μM was mixed with 10 μM siRNA inOptiMEM Low Serum Medium (Thermo Fisher). Upon complexing with siRNA,dendrimers form micelle structures with siRNA at the surface and thehydrophobic tails sequestered in the core (FIG. 16B) [19]. 14.6 μlYTZ3-15 and 10 μl siRNA were used per well of a 6-well plate. Finalconcentrations of YTZ3-15 and siRNA once added to cells were 1.75 JAMand 50 nm, respectively. Where indicated, 3.5 μM YTZ3-15 and 100 nmsiRNA were used for Ovcar8.

MSN design and delivery. Mesoporous silica nanoparticles (MSNs) weresynthesized utilizing the sol-gel method as described previously[14,20]. First, 250 mg 95% cetyltrimethylammonium bromide (CTAB) wasdissolved in 120 ml of water with 875 μl 2M sodium hydroxide solution,at 80° C. Next, 1.2 ml of 98% tetraethylorthosilicate was added. After15 min, 300 μl of 42% 3-(trihydroxysilyl) propyl methylphosphonate wasadded, and the mixture was stirred 2 hr. Particles were collected usingcentrifugation and washed with methanol. Acidic methanol was then usedto remove any remaining CTAB surfactants. Zeta potential at 50 μg/mL was43.75 mV [14]. All chemicals were obtained from Aldrich (St. Louis,Mo.). Particles were ˜120 nm in diameter, with 2.5 nm pores. Lowmolecular weight (1.8 kD branched polymer) polyethyleneimine (PEI) waselectrostatically attached to the particle surface to provide a positivechange to attract negatively charged siRNA [21]. To complex siRNA for invitro experiments, 10 μl siRNA at 10 μM was mixed with 70 ul MSNs at 500μg/ml and 20 μl water, and the mixture was incubated overnight at 4° C.on a roller. The following day, 100 μl of the MSN-siRNA complexes wasadded to each well of a 6-well plate containing 1900 pd normal medium.

Fluorescence Microscopy. To verify cell uptake of the nanoparticle-siRNAcomplexes, cells were imaged immediately before harvesting. Phase imageswere acquired, as well as fluorescent images to detectsiQ-AlexaFluor-647. Images were acquired using a Nikon TE-2000Smicroscope and SPOT Advanced software (Diagnostic Instruments, SterlingHeights, Mich.).

Confocal Microscopy. Ovcar8-IP cells were seeded into a 3.5 cm glassbottom tissue culture dish. Following attachment (24 hr), 2 ml of freshmedium replaced the old medium. Next, MSN+siQ complexes (labeled withALEXAFLUOR® 647) were added to the cells in the dish at finalconcentrations of 17.5 ng/μl (MSN) and 50 nM (siQ) and incubated for anadditional 48 hours in a tissue culture incubator. Cells were thentreated with LysoTracker Red (Thermo Fisher Scientific, Waltham, Mass.)according to the manufacturer's protocol. Confocal images were obtainedusing the Zeiss LSM700 Confocal Microscope and ZEN 2012 microscopysoftware (Zeiss A G, Oberkochen, Germany).

Western Blotting. Following siRNA treatment, cells were pelleted andlysed in RIPA buffer. Protein concentration was determined by BCA assay(Thermo Fisher). Following SDS-PAGE, protein was transferred to AmershamPVDF membrane (Genesee Scientific) using a BioRad Trans-Blot SD semi-drytransfer unit. Blots were then blocked in milk for one hour at roomtemperature or overnight at 4° C. Incubation with primary antibody tookplace for one hour at room temperature or overnight at 4° C. Antibodieswere diluted in 5% milk, with 0.1-0.2% Tween-20. Antibodies used wereTWIST 2c1a (Santa Cruz Biotechnology, Dallas, Tex.) at 1:250-1:500dilution, β-Actin, A1978 (Sigma Aldrich, St. Louis, Mo.) at1:2500-1:5000 dilution; and Horseradish Peroxidase (HRP) conjugatedanti-mouse secondary antibodies. For film-based westerns, Blue Devilfilm (Genesee Scientific) and ECL Plus chemiluminescent substrate(Thermo Fisher) were used to detect protein. For digital westerns, theSyngene Pxi4 digital blot imager and Michigan Diagnostics FemtoGlowchemilumescent substrate were used.

Sulphorhodamine B Cell Survival Assays. A2780R or Ovcar8-IP cells wereplated in 6 well plates and allowed to adhere overnight. The followingday, cells were transfected with siQ, si419, or si494 as describedabove—A2780R cells using YTZ3-15 and Ovcar8-IP cells using MSNs. After48-72 hours, cells were transferred to 96 well plates at 5,000 cells perwell and allowed to adhere overnight. The following day cells weretreated with cisplatin at a series of concentrations (cells not treatedwith cisplatin served as controls). After 72 hours, cells were fixed in10% trichloroacetic acid for 1 hour at 4° C., washed with water, anddried. Cells were stained in 0.4% sulphorhodamine B (SRB) in 1% aceticacid for 15 minutes at room temperature and then washed 3-4 times with1% acetic acid until no further color was present in the wash. Any straySRB on the walls of the wells was removed, and stained cells were driedfor 10 minutes. SRB was solubilized in 10 mM Tris base and colorintensity was quantified by absorbance at 570 nm. Each condition wasnormalized to its own untreated control.

Animal Studies. The animal studies conducted in these experiments weredone in accordance with a protocol approved by the Institutional AnimalCare and Use Committee at the City of Hope Beckman Research Institute. Atotal of 48 female NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ (NSG) mice(The Jackson Laboratory, Bar Harbor, Me.) were used. Ten week old (onaverage) mice were administered an intraperitoneal injection (IP) of2.5×10⁶ Ovcar8-IP cells in 200 μl of RPMI media. For all studies, micewere placed into four groups: MSN-siQ, MSN-siQ+cisplatin, MSN-si419H,and MSN-si419H+cisplatin (n=4 or n=8). Previous reports show no cellularuptake without MSNs¹⁴, thus a siRNA-only group was not added to thisstudy.

Bioluminescent imaging of mice (using Xenogen IVIS 100 biophotonicimaging system, STTARR, Toronto, Ontario, Canada) commenced seven daysafter injection of tumor cells in order to ensure engraftment, andcontinued once a week for four weeks. Mice were given a 100 μl IPinjection of 20 mg/ml D-Luciferin (PerkinElmer, Waltham, Mass.). Tenminutes after the D-luciferin injection, mice were anesthetized withisoflurane (2%-5%) and placed in the biophotonic imager, and images weretaken within two minutes. An alfalfa-free version of the regular rodentdiet (alfalfa-free CA-1) was administered to the siQ mice to preventautofluorescence from the regular diet.

IP injections of MSN+siRNA (siQ fluorescently labeled with ALEXAFLUOR®647 or si419H) were conducted one week after the inoculation ofOvcar8-IP cells and done once or twice per week for a total of fourweeks. Mice received 105 μl of 500 ng/μl MSN complexed with 15 μl of 10μM siRNA per week, divided into one full or two half doses (n=4 and n=8,respectively). This is equivalent to 0.04 mg/kg MSN/week. Weekly 3 mg/kgIP cisplatin injections were given starting two weeks after initialinoculation of cells.

At completion of the experiment, all animals were euthanized via CO₂asphyxiation. Both primary tumors and disseminated masses were carefullydissected from adjacent tissue and weighed. Mice treated twice weeklywith siQ only were imaged for biodistribution studies. Tumors, spleen,kidney, uterus and liver were imaged ex vivo to detect the location ofboth the Ovcar8-IP cells and AlexaFluor 647-labeled siQ without thehindrance of the skin and fur. Efficacy data is presented from micedosed once per week with MSN-siRNA.

Statistics. All in vivo data was analyzed using one-way ANOVA withcorrection for multiple comparisons, comparing all groups to MSN-siQtreatment group. Additionally, an unpaired t-test with Welch'scorrection was used to compare tumor number and weight between cisplatinalone and the combination cisplatin+MSN-si419H treatment groups. Allanalyses were done using Prism 6 software (GraphPad Software, La Jolla,Calif.). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 throughout.Power analysis demonstrates that 4 mice per group has 99% power todetect a 50% difference in means, assuming 25% standard deviation and aone-tailed test (alpha=0.05).

Results.

Dendrimer delivered siRNA knocks down TWIST in ovarian cancer celllines. We have previously showed the efficacy of our siRNAs targetingTWIST (si419 and si494, FIG. 15C) [14,15]. We used Lipofectamine 2000transfection to test si419 and si494 in A2780R cells. Both were able tocompletely inhibit TWIST expression over the course of three days (FIG.15D). In the absence of a carrier, no siRNA enters cells (FIG. 15E),therefore once siRNAs had been validated, we introduced the amphiphilicPAMAM dendrimer YTZ3-15 as a delivery vehicle. YTZ3-15 electrostaticallyattracts negatively charged siRNA using the cationic terminal amines(FIG. 16A), while the lipid tails mediate arrangement into micelles(FIG. 16B) [19]. In order to verify that dendrimers successfullydelivered siRNA into target cells, we conjugated YTZ3-15 withAlexaFluor-647 tagged siQ control siRNA. Micrographs revealed robustcell uptake of labeled siRNA in both A2780R and Ovcar8 cells (FIG. 16C).Furthermore, YTZ3-15 delivery of si419 and si494 successfully knockeddown TWIST in both lines, although knockdown in Ovcar8 was minimal andrequired double the usual siRNA-YTZ3-15 dose (FIG. 16D).

TWIST Knockdown Impacts Cisplatin Resistance. In order to determine theeffect of TWIST knockdown on cisplatin resistance, we performed asulphorhodamine B (SRB) cell survival assay. Following treatment withYTZ3-15-si494, cisplatin resistant A2780R cells were sensitized tocisplatin, with approximately one log difference in IC₅₀ (FIG. 16E).

Mesoporous Silica Nanoparticles as siRNA Delivery Vehicles. WhileYTZ3-15 treatment yielded significant TWIST knockdown in A2780R, wewanted to explore additional nanocarriers which would be capable ofmultiple functions (i.e. drug delivery and targeting via surfacemoieties) and have increased efficacy in Ovcar8. Following our recentsuccess using mesoporous silica nanoparticles (MSNs) to target TWIST invitro and in vivo in melanoma [14], we elected to apply these particlesto EOC. MSNs coated with polyethyleneimine (PEI) can carry siRNA ontheir outer surface, and contain a pore structure capable of carryingadditional cargo, such as cytotoxic drugs (FIG. 17A) [20-22]. Inaddition, MSNs can be modified with controlled release valves andtargeting moieties, further increasing their appeal [23-28]. For thepresent study, we used PEI coated MSNs without additional modificationsor drug loading. These particles were of ˜120 nm diameter with highlyuniform size (FIG. 17B). We first confirmed that PEI-coated MSNs couldeffectively deliver AlexaFluor-647 labeled siQ to both A2780R and Ovcar8cells (FIG. 17C). We found that MSNs required extended incubation withcells to produce knockdown as compared to YTZ3-15, but MSN knockdownlasted longer. With MSNs, we observed little effect at 24 hours butrobust knockdown of TWIST lasting up to one week post transfection inboth cell lines tested (FIG. 17D).

Optimization of siRNAs for In Vivo Use by Chemical Modifications. Inorder to maximize siRNA efficacy in vivo, nuclease degradation of siRNAand immune activation by siRNAs must be reduced. Immune reactions aremediated largely by toll-like receptors, which respond to varyingdegrees to different RNA sequence motifs [29]. Analysis of these motifsrevealed that si419's passenger strand, which would not be incorporatedinto RISC, contained no immune-activating sequences, and this siRNA wastherefore selected for in vivo experiments. We added additional chemicalmodifications to the si419 passenger strand to create si419 hybrid siRNA(si419H, FIG. 18A). In order to promote nuclease resistance of the siRNAduplex, 2′-O-methyl and inverted abasic ribose (iaB) modifications wereadded [30,31]. iaB also prevents the loading of the passenger strandinto RISC, effectively increasing the potency of siRNA by ensuring thatall duplexes result in binding to TWIST mRNA, improving silencing [30].

MSNs Deliver siRNA to Correct Cellular Compartment of Ovcar8-IP Cells.Ovcar8 cells were selected for further study as their genetic makeupmore closely resembles that of the typical clinical high grade serousovarian carcinoma, than the A2780 cell line family [32]. In order toimprove uniform tumor cell engraftment in mice and enable tracking ofcells in vivo, we passaged Ovcar8 cells through mice and used lentiviraltransduction to stably express an eGFP firefly luciferase fusion protein(see Methods). This line is hereafter referred to as Ovcar8-IP. Confocalmicroscopy revealed that MSN treatment of Ovcar8-IP cells resulted insiQ accumulating in the late endosomes and lysosomes of the cells, asevidenced by colocalization with LysoTracker dye (FIG. 18B). Thesestructures are located in the perinuclear space, as we have shownpreviously [14,15]. Transfection of Ovcar8-IP cells using MSNs andsi419H resulted in robust knockdown of TWIST even after 24 hours oftreatment (FIG. 18C). Furthermore, SRB assays revealed sensitization ofOvcar8-IP cells to cisplatin following MSN-419H or MSN-419 treatment(FIG. 18D). Ovcar8 cells are not as cisplatin resistant as A2780R, hencea smaller effect size compared to that seen in FIG. 16E.

MSNs Selectively Accumulate in Disseminated Ovcar8-IP Masses. In orderto determine if our MSNs targeted tumors in vivo, we performedfluorescent imaging of siQ treated mice. Ovcar8-IP cells successfullyproduced primary tumors (defined as those localized to the ovary) anddisseminated masses (FIGS. 19C-19D). This pattern of engraftmentfollowing IP administration of cells is consistent with the currentprevailing theory that the site of origin for EOC is the fallopian tubeepithelium, and that cells migrate to the ovary and peritoneal cavity,giving rise to both large ovarian tumors and widely disseminated tumorfoci [33-35]. MSNs carrying siRNA selectively penetrated and accumulatedat these primary tumors and disseminated masses, but not in any otherperitoneal tissue or organ examined (FIGS. 19E-19F). Note that a tumorfocus also appears on the liver of this control mouse, to which theMSN-siQ also homed (FIGS. 19D-19F). Tumor uptake of MSN-siQ was greaterin disseminated masses than in ovaries, but signal is visible on theprimary tumors.

Ovcar8-IP Tumor Growth is Inhibited by siTWIST and Cisplatin CombinationTherapy. Mice were treated weekly for four weeks with MSN-siQ orMSN-si419H, with or without cisplatin. After four weeks of therapy, thenegative control (MSN-siQ weekly) mice developed significant tumors andproduced disseminated masses as shown by relative bioluminescent photonflux measurements (FIGS. 20A-20B). The si419H treatment group(MSN-si419H weekly) produced relatively smaller tumors, with a 30% dropin bioluminescent signal after four weeks of therapy in comparison tothe controls. The chemotherapy only treatment group (MSN-siQ andcisplatin weekly) had a reduction rate of about 50%, whereas the si419Hwith cisplatin chemotherapy treatment group (MSN-si419H and cisplatinweekly) exhibited an almost 85% decrease in tumor burden as measured bybioluminescence (FIGS. 20A-20B). Mice treated with MSN-siRNA twiceweekly at half the dose failed to show these differences (data notshown).

Tumors collected from the MSN-si419H+cisplatin mice were significantlysmaller when compared to the tumors from the MSN-siQ control mice (FIGS.21A and 21C). Furthermore, MSN-siQ control mice produced large numbersof disseminated masses and had enlarged primary tumors (FIGS. 21A-21B)in comparison to the MSN-si419H+cisplatin treated mice that exhibited nolarge disseminated lesions. It is important to note thatcisplatin+MSN-siQ hindered tumor growth by more than 50% (FIGS.21B-21C). However, si419H and cisplatin combination therapy inhibitedOvcar8-IP tumor growth to an even greater degree, with almost a 75% dropin number of foci and tumor weight in comparison to the controls (FIGS.21B-21C). A similar trend was seen for proportion of mice developingascites, with 4/4 MSN-siQ and MSN-si419H treated mice, 2/4 cisplatintreated mice, and 1/4 combination treated mouse developing ascites.There was significant reduction in weight (p=0.00⁸⁴) and number(p=0.00⁴⁶) of disseminated masses between cisplatin+MSN-siQ andcisplatin+MSN-si419H treated mice, but this trend did not reachstatistical significance for total tumor weight (p=0.1183). This islikely the result of greater MSN-siRNA uptake by the disseminated massesthan primary tumor (FIG. 20B).

Discussion.

To our knowledge, this is the first example of silencing TWIST utilizinga nanoparticle delivery system in EOC. We demonstrate delivery andefficacy of chemically modified siRNA against TWIST in EOC; significantTWIST knockdown was achieved with a modified third generation PAMAMdendrimer and a silica nanoparticle in vitro. The delivery of our siRNAby YTZ3-15 was shown to have a profound effect on both TWIST silencingand chemoresistance in the EOC model. Silencing TWIST via the YTZ3-15dendrimer substantially increased chemosensitivity, decreasing cellsurvival by more than 50% (FIG. 16E). These data demonstrated that TWISTis a clinically significant therapeutic target.

The PEI coated MSNs developed for this study successfully deliveredsiRNA into Ovcar8-IP ovarian cancer cells in vitro (FIGS. 18B-18D). Thisdelivery also led to substantial knockdown of TWIST (FIG. 18C). Ourresults demonstrate si419H's efficacy both in vitro and in vivo.Fluorescence microscopy demonstrated appropriate localization ofMSN+Alexa-647 labeled siRNA in the lysosomes of EOC cells (FIG. 18B),which lead to successful silencing of TWIST within 24 hours and completeabsence of expression within one week (FIG. 18C). As with YTZ3-15delivery, MSN-delivered si419H sensitized EOC cells to cisplatintreatment (FIG. 18D). In vivo, delivery of TWIST siRNA leads tosignificant impediment of metastatic growth in siTWIST treated mice incombination with cisplatin, reducing tumor weight almost 80% (FIGS.21A-21C), compared to the siQ control. The observed decrease in tumorburden is mostly due to abrogated TWIST expression in EMT-mediateddisseminated masses. These findings strongly support our assertion thatTWIST is an important therapeutic target in EOC.

One of the main advantages of our system is its safety and specificity.The MSN-siRNA only localized to tumor sites and no other tissues ororgans.

The potential for these MSNs is vast due to their mesoporous nature ascompared to other dense nanoparticles, such as gold or carbon. MSNs canalso be modified with targeting moieties such as hyaluronic acid (HA).Since HA is a native ligand for CD44, which is overexpressed andcorrelates with worse prognosis in EOC [48,49], addition of HA will leadto enhanced uptake into tumors, especially in primary tumors, which inthis study showed limited siRNA uptake in vivo.

Overall, without wishing to be bound by theory, it is believe that thisdisclosure serves to demonstrate that an MSN-siRNA approach can providesignificant benefit in an EOC model, and multifunctional MSNsincorporating cytotoxic drug delivery and targeting moieties, inmultiple cancer models.

Referenced (Example 3)

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Example 4—TWIST Drives Cisplatin Resistance and Cell Survival in anOvarian Cancer Model, Via Upregulation of GAS6, L1CAM, and AktSignalling

Abstract. Epithelial ovarian cancer (EOC) is the most deadlygynaecologic malignancy due to late onset of symptoms and propensitytowards drug resistance. Epithelial-mesenchymal transition (EMT) hasbeen linked to the development of chemoresistance in other cancers, yetlittle is known regarding its role in EOC. In this study, we sought todetermine the role of the transcription factor TWIST1, a masterregulator of EMT, on cisplatin resistance in an EOC model. We createdtwo Ovcar8-derived cell lines that differed only in their TWIST1expression. TWIST1 expression led to increased tumour engraftment inmice, as well as cisplatin resistance in vitro. RNA sequencing analysisrevealed that TWIST1 expression resulted in upregulation of GAS6 andL1CAM and downregulation of HMGA2. Knockdown studies of these genesdemonstrated that loss of GAS6 or L1CAM sensitized cells to cisplatin,but that loss of HMGA2 did not give rise to chemoresistance. TWIST1, inpart via GAS6 and L1CAM, led to higher expression and activation of Aktupon cisplatin treatment, and inhibition of Akt activation sensitizedcells to cisplatin. These results suggest TWIST1- and EMT-drivenincrease in Akt activation, and thus tumour cell proliferation, as apotential mechanism of drug resistance in EOC.

Introduction. Epithelial ovarian cancer (EOC), which accounts for over90% of ovarian tumours, is the most lethal gynaecologicmalignancy.^(1,2,3). A significant challenge in the treatment of EOC isthe frequent development of tumour recurrence and chemoresistance. Weand others have previously shown that ovarian cancer stem cells (CSCs)that survive initial rounds of chemotherapy facilitate this tumourrecurrence^(4,5). A key factor in this reactivation of cancer stem cellsis TWIST1, a transcription factor that is required for normal earlymesoderm development but silenced in most adult tissues^(6,7,8). TWIST1is reactivated in many cancers, where it drives an epithelial tomesenchymal transition (EMT), leading to metastasis^(8,9). In a varietyof tumour types, TWIST1 has also been linked to angiogenesis, resistanceto apoptosis, and cancer cell stemness^(10,11,12). In ovarian cancer,TWIST1 protein is degraded in CSCs, maintaining CSCs in an epithelialstate. However, once TWIST1 protein expression persists, CSCs undergoEMT, leading to proliferation and metastasis^(5,13).

Despite its known role in activation of the stem cell pool, the directrole of TWIST1 in drug resistance in ovarian cancer is relativelyunknown. Multiple downstream TWIST1 target genes have been implicated indrug resistance, including interleukin 8 and matrix metalloproteinases 2and 9^(14,15,116). Additionally, TWIST1 has been shown to regulate Gli1,which upregulates the DNA repair protein ERCC1. ERCC1 is partiallyresponsible for the repair of cisplatin-induced DNA crosslinks^(17,18).In other tumour types, TWIST1 has been linked to resistance tocisplatin, as well as paclitaxel and doxorubicin^(19,20). While the EMTprocess as a whole has previously been correlated with drug resistancein EOC, TWIST1 itself has never been causally linked²¹. The relatedtranscription factor TWIST2 has been shown to lead to platinumresistance via Akt activation in another EOC model, but whether TWIST1can function in the same manner is unknown²². Therefore, we sought todetermine the distinct role of TWIST1 in cisplatin resistance in an EOCmodel. This study is the first to focus on the specific mechanisms bywhich TWIST1 confers cisplatin drug resistance in ovarian cancer, anovel function for a transcription factor that has previously primarilybeen only associated with tumour cell motility. Connecting TWIST1 andthe EMT process as a whole to the dual malignant functions of increasedcancerous cell proliferation and drug resistance makes it an especiallyattractive target for aggressive, drug-resistant carcinomas that requirea combination of therapeutic approaches.

Results.

Creation of Ovcar8 derived stable lines with differential TWIST1expression. The ovarian cancer cell line Ovcar8 was transfected with aviral construct encoding an enhanced GFP-firefly luciferase fusionprotein (CMV-p:EGFP-ffMuc pHIV7) to make the Ov8GFP cell line, as wehave described for previous cell line models²³. We then transfectedOv8GFP cells with either TWIST1 or sh492, a previously validated shRNAagainst TWIST1^(24,25), using the pCI-Neo G418-selectable plasmid vectorsystem. Following G418 selection of cells with stably integratedplasmid, we verified that TWIST1 was differentially expressed in the twocell lines—referred to hereafter as Ov8GFP-TWIST1 and Ov8GFP-sh492—viawestern blot (FIG. 22A). Parental Ov8GFP cells express an intermediatelevel of TWIST1, thus an empty pCI-Neo vector resulted in intermediateTWIST1 expression, showing no substantial effect on TWIST1 fromtransfection alone (FIGS. 27A-27B). Reflecting their native TWIST1expression, Ovcar8-derived lines exhibited mesenchymal morphology (FIG.28).

TWIST1 expressing cells are cisplatin resistant. We evaluated the effectof TWIST expression in response to cisplatin. Following 72 hr incubationwith cisplatin, sulphorhodamine B (SRB) cell survival assays showed thatTWIST1-overexpressing cells exhibited greater survival than TWIST1knockdown cells, normalized to untreated cells of each line (FIG. 22B).Cells transfected with empty pCI-Neo vector had intermediate survivalcompared to TWIST1 and sh492, confirming dose dependence of TWIST1 oncisplatin resistance (FIG. 22B). TWIST1 also affected the kinetics ofcell growth during cisplatin treatment. Monitoring of cell confluence at2 hr intervals showed that Ov8GFP-TWIST1 cells proliferated more rapidlythan their sh492 counterparts (compare slope of light blue vs lightgreen and dark blue vs dark green plots) when treated with 0.2 or 2 μMcisplatin (FIGS. 22C and 28).

TWIST1-expressing cells show enhanced engraftment in mice. We nextevaluated the pro-survival and proliferation phenotype ofTWIST1-overexpressing cells in tumour engraftment. We injected eitherOv8GFP-TWIST1 or Ov8GFP-sh492 cells intraperitoneally into NSG mice (n=4per group). Seven weeks after the injection, tumour burden anddistribution in these mice were evaluated via pathological examinationof haematoxylin and eosin stained tissue sections and images of theperitoneal cavity obtained at necropsy. All four mice that receivedOv8GFP-TWIST1 developed ovarian tumours were graded 4/4 by a certifiedveterinary pathologist. By contrast, only two mice given Ov8GFP-sh492exhibited ovarian tumours, only one of which was graded a 4 (Table 1).Three mice that received Ov8GFP-TWIST1 had detectable tumour masseswithin intra-abdominal organs (i.e. parenchymal tumour metastases),compared to only one of four mice in the Ov8GFP-sh492 group (Table 1).Additionally, necropsy images of mice show that mice engrafted withOv8GFP-TWIST1 had many disseminated tumour masses in the intra-abdominalperitoneal lining, which were absent in the peritoneum of the miceengrafted with Ov8GFP-sh492 (FIG. 22D). This disseminated tumourdistribution parallels the peritoneal surface carcinomatosis foundfrequently in advanced stage ovarian cancer patients. Takencollectively, these data strongly suggest a critical role for TWIST1 inpromoting survival, proliferation, and engraftment of tumour cells inthe ovaries and peritoneal space.

RNA sequencing demonstrates differential expression of GAS6, L1CAM, andHMGA2. In order to determine which downstream pathways may beresponsible for TWIST1-mediated proliferation, drug resistance, and cellsurvival, we performed RNA sequencing analysis. In addition to TWIST1itself, a total of 51 genes were found to be differentially expressedbetween Ov8GFP-TWIST1 and -sh492 (>1.5 fold difference, p<0.05), 18downregulated by TWIST1 and 33 upregulated. As expected given TWIST1'srole in EMT during development and metastasis ⁸, gene ontology (GO)terms enriched amongst TWIST1 regulated genes included Cell Movement andCell Morphology. Additional enriched GO terms included Cellular Growth/lProliferation and Cell Death and Survival. Ingenuity Pathway Analysisshowed that apoptotic and migration signalling pathways intersect atTWIST1 and its target genes, including genes identified in our RNAsequencing results. This finding suggests that TWIST1 may act to promoteboth proliferation and migration of tumour cells. A full list ofdifferentially expressed genes is given in Tables S1A-S1B following.

TABLE 1 List of differentially expressed genes output by RNA-seqanalysis. Genes are ordered according to log₂ fold change. Negative log₂indicates that gene is downregulated when TWIST1 is overexpressed andpositive log₂ indicates that gene is upregulated when TWIST1 isoverexpressed. Expression Log2 Fold Gene sh492 TWIST Change p-ValueFunction HCLS1 1.9192 0.3462 −2.4710 5.00E−05 Antigen signaling ESM119.8290 5.3762 −1.8829 5.00E−05 Proangiogenesic GGT1 7.5759 2.6660−1.5068 5.00E−05 Glutathione metabolism TIE1 4.2598 1.5563 −1.45265.00E−05 Endothelial adhesion molecule CCL20 21.4373 7.9192 −1.43675.00E−05 Downstream of IL6/NFkB/STAT3 - immune attractant ABCA1 1.79540.7054 −1.3478 5.00E−05 Cholesterol homeostasis SP4 10.2972 4.1698−1.3042 5.00E−05 Primarily neural transcription factor ARHGDIB 8.36623.4472 −1.2792 1.50E−04 Microenvironmental communication to inhibitmetastasis LSAMP 0.6617 0.2736 −1.2739 2.50E−04 Neuron growth MIR484,NDE1 36.8636 15.6657 −1.2346 1.50E−04 MicroRNA EDN1 11.9078 5.0866−1.2271 5.00E−05 Endothelin IL1A 6.1556 2.7691 −1.1525 2.00E−04Proinflammatory cytokine CTPS2 10.0673 4.8606 −1.0505 1.50E−04 CTPsynthase SERPINB2 57.3030 28.1986 −1.0230 5.00E−05 Protease inhibitor,antiapoptotic HMGA2 17.6553 8.9826 −0.9749 1.00E−04 DNA binding,chromosome condensation, DNA repair regulator LOC643201 18.1145 9.4289−0.9420 1.50E−04 Pseudogene LPHN2 34.4611 18.9748 −0.8609 1.50E−04Adhesion linked GPCR CALB1 68.4107 38.3483 −0.8351 1.00E−04 Cytosolcalcium buffering FAM129A 9.9252 17.6015 0.8265 2.50E−04 ER stressresponse, regulates translation DPYSL3 11.3916 20.8860 0.8746 5.00E−05L1CAM pathway member, putative migration function GAS6 21.2476 39.72920.9029 1.00E−04 Growth/adhesion/migration/survival. Interacts with AxlCHN1 11.6973 22.1486 0.9210 5.00E−05 Lipid stimulated GTPase GREM124.7823 47.6102 0.9420 5.00E−05 BMP inhib, proFGF signaling SHISA97.0954 13.6759 0.9467 2.00E−04 Synaptic protein EPHB1 8.8684 17.26220.9609 1.00E−04 Adhesion/migration signaling. ERK/JNK, possiblyangiogenesis RHOBTB1 1.7298 3.4910 1.0130 5.00E−05 GTPase involved withactin skeleton L1CAM 17.3307 37.0653 1.0967 5.00E−05 Cell adhesion,migration in drug resistant cancers PCSK9 4.3111 9.2437 1.1004 5.00E−05Cholesterol homeostasis OAS3 1.3659 2.9771 1.1240 1.50E−04 RNA synthesisinhibitor LOX 4.5291 10.5536 1.2204 5.00E−05 Crosslinks ECM DOK7 8.933821.3466 1.2567 5.00E−05 Neuromuscular interface ID3 17.8285 43.07581.2727 5.00E−05 Inhibitor of bHLH binding LEPREL1 8.2163 21.0238 1.35555.00E−05 Collagen assembly and linkage CAPN6 2.0987 5.5978 1.41535.00E−05 May inhibit apoptosis, promote angiogenesis FN1 225.0440620.2980 1.4628 1.50E−04 Fibronectin COL12A1 22.0742 61.1653 1.47045.00E−05 Collagen 12 alpha 1 AMIGO2 1.3070 3.9771 1.6055 5.00E−05Cell-cell communication, neg regulator of apoptosis GALNT3 0.3538 1.12921.6743 1.50E−04 Oligosaccharide biosynthesis COL4A4 1.2217 4.2168 1.78735.00E−05 Collagen alpha 4 HOXA3 0.3283 1.1875 1.8548 5.00E−05Developmental transcription factor - angiogensis and patterning ATOH80.2612 0.9560 1.8718 5.00E−05 bHLH developmental TF GDF6 0.4460 1.87932.0753 5.00E−05 Bone development - BMP signal responsive PXDNL 0.57502.4422 2.0866 5.00E−05 Oxidative stress responsive endonuclease MIR1909,REXO1 12.5434 53.5919 2.0951 5.00E−05 MicroRNA MIR5193, UBA7 0.77225.1067 2.7254 5.00E−05 MicroRNA BDKRB1 0.4423 3.1086 2.8133 5.00E−05Receptor for bradykinin, an inflammatory vasodilator LINC00452 0.23092.0640 3.1600 5.00E−05 Noncoding RNA TWIST1 20.2035 209.0290 3.37105.00E−05 EMT, angiogenesis, metastasis, stem cell phenotype MIR4324,SLC6A16 0.7451 21.5666 4.8552 5.00E−05 MicroRNA VIP 0.0000 0.4792 N/A5.00E−05 Vasodilator, also involved in survival DPT 0.0000 1.2281 N/A5.00E−05 ECM protein involved with TGFb KCNA10 0.0000 0.2646 N/A5.00E−05 Voltage gated potassium channel

As we were focused on the role of TWIST1 in drug resistance, we did notstudy any gene whose known function related only to development or cellmigration. On the contrary, on the basis of their function in regulatingcell survival, cell proliferation, and DNA repair, we selected GAS6,L1CAM, and HMGA2 for further analysis. GAS6 and L1CAM were upregulatedapproximately two fold in Ov8GFP-TWIST1 cells, while HMGA2 wasupregulated two fold in Ov8GFP-sh492 (FIG. 23A). We further verifiedthat these genes were differentially expressed in our two cell lines.Western blot analysis confirmed that L1CAM was elevated and HMGA2reduced in Ov8GFP-TWIST1 cells, as compared to Ov8GFP-sh492 (FIG. 23B).Because GAS6 is secreted from cells, its expression was confirmed byqRT-PCR rather than western. Despite variability in expression in bothOv8GFP-TWIST1 and -sh492 cells, TWIST1 expressing cells had two-foldhigher levels of GAS6 mRNA on average (FIG. 23C). We also found thattumours from mice given Ov8GFP-TWIST1 cells showed uniform IHC stainingfor L1CAM. Tumours from sh492 mice were heterogeneous, with areas inwhich staining was entirely absent (FIG. 29).

We next knocked down each of these three genes to observe theirindividual effects on TWIST1-driven cell survival. qRT-PCR showed thatsiRNA against L1CAM and GAS6 produced 46% and 90% knockdown of theirtarget mRNAs, respectively, in Ov8GFP-TWIST1 cells compared tonon-targeting control siRNA (siQ) (FIGS. 24A-24B). An siRNA pool againstHMGA2 reduced HMGA2 mRNA levels by 91% on average in Ov8GFP-sh492 cells(FIG. 24C). Knockdown of L1CAM and HMGA2 by their respective siRNAsequences was also confirmed at the protein level via western blot (FIG.24D).

HMGA2 knockdown does not confer cisplatin resistance. As HMGA2 is anegative regulator of ERCC1²⁶, we hypothesized that knockdown of HMGA2might upregulate the DNA repair pathway responsible for the repair ofthe DNA crosslinks caused by cisplatin. Thus, we expected that HMGA2knockdown cells would show enhanced cisplatin resistance. However, anSRB cell survival assay showed that HMGA2 knockdown had no impact on theproportion of Ov8GFP-sh492 cells able to survive cisplatin treatment(FIG. 24E). This may be due to the redundancy of DNA repair pathways orthe compensatory activation of ERCC1 by additional factors; however,further studies are needed to determine if this is truly the case.

Knockdown of GAS6 or L1CAM sensitizes cells to cisplatin. We alsohypothesized that knockdown of GAS6 or L1CAM might sensitize cells tocisplatin due to abrogated survival signalling downstream from thesefactors. In order to test this hypothesis, we performed an SRB assay onOv8GFP-TWIST1 cells treated with siQ or siRNA pools against GAS6 orL1CAM. We found that knockdown of either gene was able to sensitizecells to cisplatin, with L1CAM knockdown reducing cell survival by up to20% (FIG. 24F).

TWIST1, GAS6, and L1CAM upregulate expression and phosphorylation of Aktin response to cisplatin. Given that both GAS6 and L1CAM have beenlinked to Akt signalling^(27, 28), and that TWIST2-mediated activationof Akt has been previously implicated in acquired cisplatinresistance²², we hypothesized that Akt may also be a key factordownstream from TWIST, GAS6, and L1CAM. We also hypothesized thatknockdown of GAS6 or L1CAM in TWIST1 overexpressing cells could inhibitupregulation and activation of Akt.

Following treatment of cells with siQ control siRNA or pooled siRNAsagainst GAS6 or L1CAM, western blotting of total Akt showed that whileOv8GFP-TWIST1 cells have lower initial Akt expression compared toOvGFP-sh492, continued exposure to 5 μM cisplatin led to a 150% increasein Akt levels in Ov8GFP-TWIST1 cells over the course of 24 hr (FIG.25A). In Ov8GFP-sh492 cells, total Akt levels remain relatively constantover 24 hr of cisplatin exposure (FIG. 25A). Interestingly, theproportion of Akt in its active form (i.e. phosphorylated at Ser 473)increases 128% over the course of 24 hr in Ov8GFP-TWIST1 cells, evenwhen normalized to total Akt expression at each time point (FIG. 25B).Conversely, OvGFP-sh492 cells show a 63% reduction in phosphorylated Aktover the same 24 hr period (FIG. 25B). The pattern of Akt activation inthese cell lines mirrors the activation of TWIST1 itself inOv8GFP-TWIST1 cells over 24 hours of cisplatin treatment, which isabsent in sh492 cells (FIG. 25C).

Western blotting also revealed that knockdown of either GAS6 or L1CAMcould partially prevent Akt upregulation, as It resulted in largelyconstant Akt levels over time (FIG. 25A). Similarly, knockdown of eitherGAS6 or L1CAM produced levels of Akt phosphorylation intermediatebetween those seen in Ov8GFP-TWIST1 and OvGFP-sh492 cells treated withsiQ control. GAS6 knockdown kept the proportion of active Akt relativelyconstant, while loss of L1CAM led to increasing Akt activation at eachtime point, but only 53% over 24 hr, as compared to 128% for siQ treatedOv8GFP-TWIST1 cells (FIG. 25B).

Inhibition of Akt activation sensitizes cells to cisplatin. In order toconfirm that Akt mediates cisplatin resistance downstream of TWIST1 inour system, we treated Ov8GFP-TWIST1 cells with either cisplatin alone,or cisplatin plus the PI3K inhibitor LY294002, which prevents thephosphorylation of Akt by PI3K. Cells treated with the combinationexhibited 15% greater cell death at 5 μM cisplatin and 25% greater celldeath at 10 μM, compared to those treated with cisplatin alone,confirming that loss of Akt activation leads to cisplatin sensitivity inour model (FIG. 25D). This link between TWIST1 and Akt function,combined with the tumour engraftment data presented in FIGS. 22A-22D,suggests that TWIST1-mediated cisplatin resistance may be a part of anoverall increase in cell growth and proliferation signalling.

Discussion.

Epithelial ovarian cancer is characterized by tumours that are widelydisseminated throughout the peritoneal cavities of patients and thathave a high tendency for recurrence. Both of these unwanted phenotypesare made possible by the presence of cancer stem cells. As these cellsare quiescent, drugs such as paclitaxel and cisplatin that targetrapidly proliferating cells have little efficacy. Unfortunately once thebulk of the tumour mass has been eliminated by surgery and chemotherapy,CSCs drive cancer recurrence by re-entering the cell cycle anddifferentiating. We have previously shown that during this process ofre-entry, the CSCs lose expression of CD44 and MyD88 and acquiremesenchymal characteristics^(5,13) due to the persistence of TWIST1protein.

A growing body of studies link TWIST1 to many cancer processes outsideof its traditionally studied roles in cell migration and metastasis.This current study examines additional cancer phenotypic impacts ofTWIST1 in the context of differentiated EOC cells. We sought todetermine the role TWIST1 plays in the acquisition of drug resistance inrecurrent tumour cells. Prior studies have linked TWIST1 and the relatedprotein TWIST2 to drug resistance in multiple tumour types, includingovarian, but the specific mechanism by which TWIST1 drives resistance inEOC is not well understood. To elucidate this mechanism, we created apair of cell lines in the Ovcar8 background which differed in expressionof TWIST1. We then employed SRB cell survival assays and IncuCyte cellgrowth studies to monitor the effects of TWIST1 and its target genes,providing both a static and dynamic measurement of cell proliferation,and using assays well suited to the Ovcar8 line.

In our EOC model, we found that sustained TWIST overexpression in EOCcells with a mesenchymal phenotype led to enhanced cell survival andproliferation, both in the in vivo tumour engraftment assays and in thepresence of cisplatin in vitro (FIGS. 22A-22D). RNA-sequencing analysisof TWIST1-overexpressing and TWIST1-knockdown cells revealed 51significantly differentially expressed genes. As expected given TWIST1'swell documented role in modulating cell migration and cell-extracellularmatrix interactions, several of the genes identified related to theseprocesses (Tab Si). However, a number of genes also related to cellsurvival and proliferation signalling. Of these, we selected GAS6,L1CAM, and HMGA2 for further study. We verified differential expressionof these genes in our Ov8GFP-TWIST1 and -sh492 cell lines (FIGS.23A-23B), and validated a pool of siRNAs against each (FIGS. 24A-24F).HMGA2 has previously been linked to TWIST1, but in metastatic breastcancer, in which TWIST1 and HMGA2 are both targets of microRNAmiR-33b²⁹. HMGA2 is a negative regulator of the nucleotide excisionrepair (NER) protein ERCC1, which is involved in the repair ofplatinum-induced DNA crosslinks^(18,26). We therefore hypothesized thatknockdown of HMGA2 would allow for cisplatin-induced upregulation of NERas previously reported ¹⁸, and that HMGA2 knockdown in Ov8GFP-sh492cells would give rise to cisplatin resistance. However, we found thatloss of HMGA2 did not have any effect on cell survival in response tocisplatin (FIG. 24E). This may be due to redundancy of DNA repairsignalling, as multiple factors will likely regulate NER, includingGli1¹⁸, and knockdown of a single regulatory protein may not besufficient to impact NER function.

We next determined the impact of GAS6 and L1CAM in the response tocisplatin. We found that knockdown of either gene was able to sensitizeTWIST1 overexpressing cells to cisplatin (FIG. 24F).

We next sought to determine if TWIST1, along with GAS6 and L1CAM, wasacting via Akt. We found that expression of TWIST1 led to an increase inAkt expression and activation over the course of 24 hr of cisplatintreatment in vitro, while TWIST1 knockdown led to a decrease in Aktactivity (FIGS. 25A-25B). Knockdown of GAS6 or L1CAM partially preventedAkt activation in Ov8GFP-TWIST1 cells (FIGS. 25A-25B). As expected,inhibition of Akt activation by the PI3K inhibitor LY294002substantially sensitized OV8GFP-TWIST1 cells to cisplatin, even aftercorrecting for the anti-proliferative effects of the inhibitor (FIG.25C).

Taken together, our data suggest a model where TWIST1-mediatedupregulation of L1CAM expression and GAS6/Ax1 signalling lead to higherthroughput of Akt signalling. This increase in proliferation gives riseto greater cell survival during tumour cell engraftment assays. Thismodel also suggests that TWIST1-mediated drug resistance is a result ofincreased proliferation, rather than direct inhibition of cisplatinactivity by DNA repair proteins or upregulation of drug efflux, forexample by downregulation of HMGA2 in TWIST1 overexpressing cells (FIG.26). This is the first study on the role TWIST1 plays in acquired drugresistance in ovarian cancer.

Methods.

Cell lines. Ovcar8 cells were obtained from ATCC, and engineered tostably express a GFP-firefly luciferase fusion protein, using theCMV-p:EGFP-ffluc pHIV7 vector (a gift from Christine Brown at City ofHope, as has been described previously²³) to make the Ov8GFP line. TheTWIST1 gene or an shRNA targeting TWIST1, sh492, were cloned into thepCI-Neo vector from Promega (Madison, Wis.). Empty pCI-Neo vector wasused as control. Lipofectamine 2000 (Thermo Fisher, Waltham, Mass.) wasused to transfect the vectors into Ov8GFP cells and cells were treatedwith 0.8 mg/mL G418 (Sigma Aldrich, St. Louis, Mo.) to select for stableplasmid integration. Resulting cell lines are herein referred to asOv8GFP-TWIST1, Ov8GFP-pCI-Neo, and Ov8GFP-sh492. sh492 includes ananti-TWIST siRNA sequence of 5′-GCGACGAGCUGGACUCCAA-3′ (SEQ ID NO: 34),and is used for establishing stable cell lines using adenoviraltransduction.

Cell culture. All cells were grown in RPMI 1640 medium (GeneseeScientific, San Diego, Calif.) supplemented with 10% foetal bovine serumand 1% penicillin/streptomycin, in a tissue culture incubatormaintaining 37° C., 5% CO₂, 90% humidity. Ov8GFP-TWIST1, Ov8GFP-pCI-Neo,and Ov8GFP-sh492 were grown in 0.4 mg/mL G418 to maintainTWIST1/pCI-Neo/sh492 plasmid integration. Cells were passaged every 2-4days. Confluent cells were washed with PBS, detached with 0.25%trypsin-EDTA (Genesee Scientific), and transferred to new dishes.

Gene knockdown. Small interfering RNA (siRNA) was used for knockdown ofGAS6, L1CAM, and HMGA2. Lipofectamine 2000 was used to transfect siRNAinto cells in OptiMEM medium (Thermo Fisher). Medium was changed tonormal medium after 4 hr or on the following day. Non-targeting controlsiRNA, siQ, was AllStars Negative Control siRNA from Qiagen (Valencia,Calif.). Pooled siRNAs against GAS6, L1CAM, and HMGA2 were obtained fromSanta Cruz Biotechnology (Dallas, Tex.; item numbers sc-35450, sc-43172,and sc-37994, respectively).

qRT-PCR. Total RNA was isolated from pelleted cells using the RNeasyPlus kit from Qiagen according to the manufacturer's protocol. cDNA wasreverse transcribed using the iScript cDNA Synthesis kit from Bio-Rad(Hercules, Calif.). Real time PCR was run either on an AppliedBiosystems StepOnePlus machine using SYBR Select Master Mix (LifeTechnologies, Carlsbad, Calif.) or on a Bio-Rad iQ5 system using SYBRmaster mix from Kapa Biosystems (Wilmington, Mass.) in 20 μL reactions,in triplicate. Melt curves were obtained for all reactions. β-Actin wasused as the endogenous control. Expression was determined using the2^(−ΔΔCt) method. Primers used were:

GAS6 Fwd (SEQ ID NO: 26) 5′-CTGCATCAACAAGTATGGGTCTCCGT-3′, GAS6 Rev(SEQ ID NO: 27) 5′-GTTCTCCTGGCTGCATTCGTTGA-3′, HMGA2 Fwd (SEQ ID NO: 28)5′-CAGCGCCTCAGAAGAGAGGACG-3′, HMGA2 Rev (SEQ ID NO: 29)5′-CCGTTTTTCTCCAGTGGCTTCTGCT-3′, L1CAM Fwd (SEQ ID NO: 30)5′-GCAGCAAGGGCGGCAAATACTCA-3′, L1CAM Rev (SEQ ID NO: 31)5′-CTTGATGTCCCCGTTGAGCGAT-3′, β-Actin Fwd (SEQ ID NO: 32)5′-CCGCAAAGACCTGTACGCCAAC-3′, β-Actin Rev (SEQ ID NO: 33)5′-CCAGGGCAGTGATCTCCTTCTG-3′.

Western blotting. cells were pelleted and washed once with phosphatebuffered saline (PBS), then lysed in RIPA buffer. Protein concentrationwas determined using a BCA assay (Thermo Fisher). Equal masses ofprotein were run on 4% stacking, 10% resolving polyacrylamide gels, andthen transferred to PVDF membrane (GE Healthcare Bio-Sciences,Pittsburgh, Pa.) using the Trans-Blot SD Semi-Dry Transfer Cell(Bio-Rad). Membranes were blocked in dry milk dissolved in PBS withshaking for 1 hr at room temperature or overnight at 4° C. 3% BSA wasused for blocking prior to select pAkt blots to reduce backgroundsignal. Primary and secondary antibodies were diluted as indicated belowin 5% dry milk in PBS with 0.1-0.2% Tween-20. Incubation was performedat room temperature for one hr or overnight at 4° C. for primary, and atroom temperature for one hr for secondary. Each antibody incubation wasfollowed by five 5 min washes in PBS with 0.1% Tween-20. FemtoGlow HRPsubstrate (Michigan Diagnostics, Royal Oak, Mich.) and the Pxi4chemiluminescent imager (Syngene, Frederick, Md.) were used to acquiredigital images. ECL 2 (Thermo Fisher) and Blue Devil Film (GeneseeScientific) were used to acquire film images. FemtoGlow was diluted6-fold in water for imaging of actin bands. Membranes were strippedusing Restore Western Blot Stripping Buffer (Thermo Fisher), rinsedtwice in PBS, and the process was repeated for each protein tested.Antibodies used were TWIST1 (TWIST 2c1a, Santa Cruz Biotechnologysc-81417, 1:500), β-Actin (Sigma Aldrich A1978, 1:5,000), Phospho Akt(Ser473) (Cell Signaling Technology 9271, 1:1000), Akt (Cell SignalingTechnology 9272, 1:1000), HMGA2 (HMGI-C 2421C6a, Santa CruzBiotechnology sc-130024, 1:500), and L1CAM (NCAM-L1 D5, Santa CruzBiotechnology sc-374046, 1:1000). For select digital and film westerns,bands were quantified using GeneTools software from Syngene or ImageStudio Lite from LiCor (Lincoln, Nebr.), respectively, and normalized toJ3-Actin.

RNA sequencing analysis. RNA from biological replicates of Ov8GFP-sh492and -TWIST1 was obtained as described for qRT-PCR. Quality was verifiedby absorption spectra using a NanoDrop 1000 spectrophotometer (ThermoFisher). RNA sequencing of two samples per cell line was performed intriplicate using the Illumina Hi-seq platform by the IntegrativeGenomics Core facility at City of Hope. Data were analysed using theonline Galaxy platform. Pipeline consisted of the following algorithms:Tophat (for alignment of sequenced fragments to the human genome),Cufflinks (for the assembly of aligned fragments into transcripts),Cuffmerge (for the merging of several Cufflink assemblies into a singlefile), and Cuffdiff (for determining differences in expression using atwo-sample t-test). Ingenuity Pathway Analysis was used to buildrelationships and potential links to canonical signalling pathways basedon previously published publications.

Sulphorhodamine B assays. Cells were plated at 5,000 per well in normalmedium in 96 well plates (100 μL/well), n=6 per drug concentration, percondition. Cells were allowed to adhere overnight, and then cisplatinwas added. Cisplatin was prepared at 2× concentration in 100 μL normalmedium, and then added to cells to yield 200 μL at 1×. Following 3 dayincubation with cisplatin, medium was removed and cells were fixed with10% trichloroacetic acid (TCA, 100 μL/well) at 4° C. for 1 hr. TCA wasthen removed and wells were rinsed with 200 μl water and allowed to airdry 10 min. Next, cells were incubated in 0.4% sulphorhodamine B (SRB)in 1% acetic acid at room temperature for 15 minutes, after which dyewas discarded and wells were rinsed 3-4 times with 1% acetic acid untilwash showed no further colour. Plates were air dried and excess SRBadhered to the walls of the wells was removed with a cotton swab.Finally, SRB was solubilized in 10 mM Tris base (200 μL/well) andabsorbance was measured at 570 nm on a SpectraMax Plus plate reader(Molecular Devices, Sunnyvale, Calif.). Readings for each condition werenormalized to untreated wells from the same condition. Cisplatinconcentrations were adjusted to fit experimental demands. Lipofectaminetransfected cells were more sensitive to cisplatin, thus a reducedcisplatin concentration across all conditions was required. For Aktstudies, the PI3K inhibitor LY294002 was used to inhibit Akt activation(FIGS. 25A-25D), and was obtained from Cell Signaling Technology(Danvers, Mass.).

Real-Time monitoring of cell confluence. In order to determine theeffects of TWIST1 expression and cisplatin on the kinetics of cellgrowth, the IncuCyte ZOOM system (Essen BioScience, Ann Arbor, Mich.)was used as described previously [4, 50, 51]. Briefly, Ov8GFP-TWIST1 andOv8GFP-sh492 cells were plated at 4,000 cells/well of a 96 well plate inquadruplicate overnight in normal medium. The following day, they weretreated with 0.2 or 2 μM cisplatin (Teva Pharmaceuticals USA,Sellersville, P A) and imaged every two hr for 74 hr to determine cellconfluence over time.

In vivo tumorigenesis study. To determine the effects of TWIST1 ontumour engraftment and proliferation in vivo, 3.2 million Ov8GFP-TWIST1or Ov8GFP-sh492 cells were injected intraperitoneally into femaleNOD.Cg-Prkdc^(scid)Il2rg^(tm1Wj1)/SzJ (NSG) mice (The JacksonLaboratory, Bar Harbor, Me.). Four mice received each cell line. Tumourswere allowed to grow for seven weeks, and tumour burden was evaluated atnecropsy. Images were taken of mice, and peritoneal organs wereharvested and fixed in formalin for evaluation by a board-certifiedveterinary pathologist with over 30 years of experience in pathology ofexperimental mouse models. Evaluation was done without knowledge oftreatment group or experimental design. The amount of tumour growthwithin a tissue was graded using a progressive, semi-quantitative,tiered scale of 0-4, where 0=no tumour growth and 4=major portion of thetissue occupied by tumour growth. This study was conducted in accordancewith a protocol approved by the Institutional Animal Care and UseCommittee at the City of Hope Beckman Research Institute (Protocol no.15002, approved 22 Mar. 2016). Care was taken to minimise the number ofmice used and the pain and discomfort of mice in the study. Results ofpathology analyses are tabulated in Table 2 following.

TABLE 2 Results of pathology analysis. TWIST1 overexpressing cells gaverise to large ovarian tumours in 4/4 mice, whereas sh492 expressingcells gave rise to tumours in 2/4 mice, with only one matching theseverity seen in TWIST1 tumours (1/4 sh492 scored 4 vs 4/4 TWIST1 scored4). 3/4 mice receiving TWIST1- expressing cells developed a metastaticlesion in their liver or spleen, compared to 1/4 sh492 mice. A, B, C,and D refer to individual mice. “0” reflects a tumour score of 0, while“—” denotes no sample collected. Tumour Score Mouse Liver Uterus OvaryKidney Spleen sh492 A 0 0 4 0 0 sh492 B 1 0 2 0 0 sh492 C 0 0 0 0 0sh492 D 0 — — 0 0 TWIST1 A 0 0 4 0 0 TWIST1 B 0 — 4 0 1 TWIST1 C 1 0 4 00 TWIST1 D 1 0 4 0 0

Statistics. Statistical significance between conditions forcytotomoeity, assays was determined by a series of unpaired Studentt-tests comparing TWIST1 to sh492 or comparing gene knockdown conditionsto siQ control. The Holm-Sidak method was used to correct for multiplecomparisons. No assumption of equal standard deviation was made.Knockdown of HMGA2 and L1CAM protein was analysed using paired,one-sided t-tests. All calculations were done using Prism 6 (GraphPadSoftware, La Jolla, Calif.). Asterisks denote statistical significance(p<0.05). Exact p-values and error bar parameters are given in figurelegends. OneStep qPCR data, RNA-seq output, and slopes of IncuCytegraphs are not open to statistical analysis by our software, and sotrends are numerically described in the text for these experiments.

References (Example 4)

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What is claimed is:
 1. A composition comprising a TWIST signalinginhibitor bound to a delivery vehicle.
 2. The composition of claim 1,wherein said TWIST signaling inhibitor is an siRNA inhibitor or a smallmolecule inhibitor.
 3. The composition of claim 1, wherein said deliveryvehicle is a nanoparticle or a lipid particle.
 4. The composition ofclaim 2, wherein said siRNA inhibitor is an anti-TWIST siRNA.
 5. Thecomposition of claim 4, wherein said anti-TWIST siRNA comprises asequence of any one of SEQ ID Nos: 1-10, or a complementary sequencethereof.
 6. The composition of claim 5, wherein said sequence comprisesa nucleic acid modification.
 7. The composition of claim 6, wherein saidnucleic acid modification is a 2′-O-methyluracil or inverted abasicdeoxyribose.
 8. The composition of claim 6, wherein said modification isa 2-thio-deoxyuracil.
 9. The composition of claim 3, wherein saidnanoparticle is a mesoporous silica nanoparticle (MSN).
 10. Thecomposition of claim 9, wherein said MSN is bound to polyethyleneimine(PEI).
 11. The composition of claim 3, wherein said nanoparticle is adendrimer-based nanoparticle.
 12. The composition of claim 11, whereinsaid dendrimer-based nanoparticle is YTX3-15.
 13. The composition ofclaim 1, wherein said TWIST signaling inhibitor is an inhibitor ofgrowth arrest-specific 6 (GAS6), L1 cell adhesion molecule (L1CAM) or anAkt signaling factor.
 14. The composition of claim 13, wherein said Aktsignaling factor is phosphatidylinositol 3-kinase (PI3K) or proteinkinase B (Akt).
 15. The composition of claim 1, further comprising apharmaceutically acceptable excipient to form a pharmaceuticalcomposition.